Methods for treating patients with adenoviral vectors

ABSTRACT

The present invention addresses the need to improve the yields of viral vectors when grown in cell culture systems. In particular, it has been demonstrated that for adenovirus, the use of low-medium perfusion rates in an attached cell culture system provides for improved yields. In other embodiments, the inventors have shown that there is improved Ad-p53 production witrh cells grown in serum-free conditions, and in particular in serum-free suspension culture. Also important to the increase of yields is the use of detergent lysis. Combination of these aspects of the invention permits purification of virus by a single chromatography step that results in purified virus of the same quality as preparations from double CsCl banding using an ultracentrifuge.

BACKGROUND OF THE INVENTION

[0001] The present application is a continuation-in-part of co-pendingU.S. Provisional Patent Application Ser. No. 60/031,329 filed Nov. 20,1997. The entire text of the above-referenced disclosure is specificallyincorporated by reference herein without disclaimer.

[0002] 1. Field of the Invention

[0003] The present invention relates generally to the fields of cellculture and virus production. More particularly, it concerns improvedmethods for the culturing of mammalian cells, infection of those cellswith adenovirus and the production of infectious adenovirus particlestherefrom.

[0004] 2. Description of Related Art

[0005] Adenoviral vectors, which express therapeutic proteins, arecurrently being evaluated in the clinic for the treatment of a varietyof cancer indications, including lung and head and neck cancers. As theclinical trials progress, the demand for clinical grade adenoviralvectors is increasing dramatically. The projected annual demand for a300 patient clinical trial could reach approximately 6×10¹⁴ PFU.

[0006] Traditionally, adenoviruses are produced in commerciallyavailable tissue culture flasks or “cellfactories.” Virus infected cellsare harvested and freeze-thawed to release the viruses from the cells inthe form of crude cell lysate. The produced crude cell lysate (CCL) isthen purified by double CsCl gradient ultracentrifugation. The typicallyreported virus yield from 100 single tray cellfactories is about 6×10¹²PFU. Clearly, it becomes unfeasible to produce the required amount ofvirus using this traditional process. New scaleable and validatableproduction and purification processes have to be developed to meet theincreasing demand.

[0007] The purification throughput of CsCl gradient ultracentrifugationis so limited that it cannot meet the demand for adenoviral vectors forgene therapy applications. Therefore, in order to achieve large scaleadenoviral vector production, purification methods other than CsClgradient ultracentrifugation have to be developed. Reports on thechromatographic purification of viruses are very limited, despite thewide application of chromatography for the purification of recombinantproteins. Size exclusion, ion exchange and affinity chromatography havebeen evaluated for the purification of retroviruses, tick-borneencephalitis virus, and plant viruses with varying degrees of success(Crooks, et al., 1990; Aboud, et al., 1982; McGrath et al., 1978, Smithand Lee, 1978; O'Neil and Balkovic, 1993). Even less research has beendone on the chromatographic purification of adenovirus. This lack ofresearch activity may be partially attributable to the existence of theeffective, albeit non-scalable, CsCl gradient ultracentrifugationpurification method for adenoviruses.

[0008] Recently, Huyghe et al. (1996) reported adenoviral vectorpurification using ion exchange chromatography in conjunction with metalchelate affinity chromatography. Virus purity similar to that from CsClgradient ultracentrifugation was reported. Unfortunately, only 23% ofvirus was recovered after the double column purification process.Process factors that contribute to this low virus recovery are thefreeze/thaw step utilized by the authors to lyse cells in order torelease the virus from the cells and the two column purificationprocedure.

[0009] Clearly, there is a demand for an effective and scaleable methodof adenoviral vector production that will recover a high yield ofproduct to meet the ever increasing demand for such products.

SUMMARY OF THE INVENTION

[0010] The present invention describes a new process for the productionand purification of adenovirus. This new production process offers notonly scalability and validatability but also virus purity comparable tothat achieved using CsCl gradient ultracentrifugation.

[0011] Thus the present invention provides a method for producing anadenovirus comprising growing host cells in media at a low perfusionrate, infecting the host cells with an adenovirus, harvesting and lysingthe host cells to produce a crude cell lysate, concentrating the crudecell lysate, exchanging buffer of crude cell lysate, and reducing theconcentration of contaminating nucleic acids in the crude cell lysate.

[0012] In particular embodiments, the method further comprises isolatingan adenoviral particle from the lysate using chromatography. In certainembodiments, the isolating consists essentially of a singlechromatography step. In other embodiments, the chromatography step ision exchange chromatography. In particularly preferred embodiments, theion exchange chromatography is carried out at a pH range of betweenabout 7.0 and about 10.0. In more preferred embodiments, the ionexchange chromatography is anion exchange chromatography. In certainembodiments the anion exchange chromatography utilizes DEAE, TMAE, QAE,or PEI. In other preferred embodiments, the anion exchangechromatography utilizes Toyopearl Super Q 650M, MonoQ, Source Q orFractogel TMAE.

[0013] In certain embodiments of the present invention the glucoseconcentration in the media is maintained between about 0.7 and about 1.7g/L. In certain other embodiments, the exchanging buffer involves adiafiltration step.

[0014] In preferred embodiments of the present invention, the adenoviruscomprises an adenoviral vector encoding an exogenous gene construct. Incertain such embodiments, the gene construct is operatively linked to apromoter. In particular embodiments, the promoter is SV40 IE, RSV LTR,β-actin or CMV IF adenovirus major late, polyoma F9-1, or tyrosinase. Inparticular embodiments of the present invention, the adenovirus is areplication-incompetent adenovirus. In other embodiments, the adenovirusis lacking at least a portion of the El-region. In certain aspects, theadenovirus is lacking at least a portion of the E1A and/or E1B region.In other embodiments, the host cells are capable of complementingreplication. In particularly preferred embodiments, the host cells are293 cells.

[0015] In preferred a embodiment of the present invention it iscontemplated that the exogenous gene construct encodes a therapeuticgene. For example, the therapeutic gene may encode antisense ras,antisense myc, antisense raf, antisense erb, antisense src, antisensefins, antisense jun, antisense trk antisense ret, antisense gsp,antisense hst, antisense bcl antisense abl, Rb, CFTR, p16, p21, p27,p57, p73, C-CAM, APC, CTS-1, zac1, scFV ras, DCC, NF-1, NF-2, WT-1,MEN-I, MEN-II, BRCA1, VHL, MMAC1, FCC, MCC, BRCA2, IL-I, IL-2, IL-3,IL4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, GM-CSF G-CSF,thymidine kinase or p53.

[0016] In certain aspects of the present invention, the cells may beharvested and lysed ex situ using a hypotonic solution, hypertonicsolution, freeze-thaw, sonication, impinging jet, microfluidization or adetergent. In other aspects, the cells are harvested and lysed in situusing a hypotonic solution, hypertonic solution, or a detergent. As usedherein the term “in situ” refers to the cells being located within thetissue culture apparatus for example CellCube™ and “ex situ” refers tothe cells being removed from the tissue culture apparatus.

[0017] In particular embodiments, the cells are lysed and harvestedusing detergent. In preferred embodiments the detergent may be Thesit®,NP40®, Tween-20®, Brij-58®, Triton X®-100 or octyl glucoside. In otheraspects of the present invention lysis is achieved through autolysis ofinfected cells. In certain other aspects of the present invention thecell lysate is treated with Benzonase®, or Pulmozyme®.

[0018] In particular embodiments, the method further comprises aconcentration step employing membrane filtration. In particularembodiments, the filtration is tangential flow filtration. In preferredembodiments, the filtration may utilize a 100 to 300K NMWC, regeneratedcellulose, or polyether sulfone membrane.

[0019] The present invention also provides an adenovirus producedaccording to a process comprising the steps of growing host cells inmedia at a low perfusion rate, infecting the host cells with anadenovirus, harvesting and lysing the host cells to produce a crude celllysate, concentrating the crude cell lysate, exchanging buffer of crudecell lysate, and reducing the concentration of contaminating nucleicacids in the crude cell lysate.

[0020] Other aspects of the present invention provide a method for thepurification of an adenovirus comprising growing host cells, infectingthe host cells with an adenovirus, harvesting and lysing the host cellsby contacting the cells with a detergent to produce a crude cell lysate,concentrating the crude cell lysate, exchanging buffer of crude celllysate, and reducing the concentration of contaminating nucleic acids inthe crude cell lysate.

[0021] In particular embodiments, the detergent may be Thesit®, NP-40®,Tween-20®, Brij-58®, Triton X-100® or octyl glucoside. In moreparticular embodiments the detergent is present in the lysis solution ata concentration of about 1% (w/v).

[0022] In other aspects of the present invention there is provided anadenovirus produced according to a process comprising the steps ofgrowing host cells, infecting the host cells with an adenovirus,harvesting and lysing the host cells by contacting the cells with adetergent to produce a crude cell lysate, concentrating the crude celllysate, exchanging buffer of crude cell lysate, and reducing theconcentration of contaminating nucleic acids in the crude cell lysate.

[0023] In yet another embodiment, the present invention provides amethod for the purification of an adenovirus comprising the steps ofgrowing host cells in serum-free media; infecting said host cells withan adenovirus; harvesting and lysing said host cells to produce a crudecell lysate; concentrating said crude cell lysate; exchanging buffer ofcrude cell lysate, and reducing the concentration of contaminatingnucleic acids in said crude cell lysate. In preferred embodiments, thecells may be grown independently as a cell suspension culture or as ananchorage-dependent culture.

[0024] In particular embodiments, the host cells are adapted for growthin serum-free media. In more preferred embodiments, the adaptation forgrowth in serun-free media comprises a sequential decrease in the fetalbovine serum content of the growth media. More particularly, theserum-free media comprises a fetal bovine serum content of less than0.03% v/v.

[0025] In other embodiments, the method further comprises isolating anadenoviral particle from said lysate using chromatography. In preferredembodiments, the isolating consists essentially of a singlechromatography step. More particularly, the chromatography step is ionexchange chromatography.

[0026] Also contemplated by the present invention is an adenovirusproduced according to a process comprising the steps of growing hostcells in serum-free media; infecting said host cells with an adenovirus;harvesting and lysing said host cells to produce a crude cell lysate;concentrating said crude cell lysate; exchanging buffer of crude celllysate; and reducing the concentration of contaminating nucleic acids insaid crude cell lysate.

[0027] The present invention further provides a 293 host cell adaptedfor growth in serum-free media In certain aspects, the adaptation forgrowth in serum-free media comprises a sequential decrease in the fetalbovine serum content of the growth media. In particular embodiments, thecell is adapted for growth in suspension culture. In particularembodiments, the cells of the present invention are designated IT293SFcells. These cells were deposited with the American Tissue CultureCollection (ATCC) in order to meet the requirements of the BudapestTreaty on the international recognition of deposits of microorganismsfor the purposes of patent procedure. The cells were deposited by Dr.Shuyuan Zhang on behalf of Introgen Therapeutics, Inc. (Houston, Tex.),on Nov. 17, 1997. IT293SF cell line is derived from an adaptation of 293cell line into serum free suspension culture as described herein. Thecells may be cultured in IS 293 serum-free media (Irvine Scientific.Santa -Ana, Calif.) supplemented with 100 mg/L heparin and 0.1% pluronicF-68, and are permissive to human adenovirus infection.

[0028] Other objects, features and advantages of the present inventionwill become apparent from the following detailed description. It shouldbe understood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The following drawings form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

[0030]FIG. 1A and FIG. 1B. HPLC profiles of the viral solutions fromproduction runs using medium perfusion rates characterized as “high”(FIG. 1A) and “low” (FIG. 1B).

[0031]FIG. 2. The HPLC profile of crude cell lysate (CCL) from CellCube™(solid line A₂₆₀; dotted line A₂₈₀).

[0032]FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D and FIG. 3E. The HPLC profilesof lysis solutions from CellCube™ using different detergents. FIG. 3AThesit®. FIG. 3B Triton®X-100. FIG. 3C. NP-40®. FIG. 3D. Brij®80. FIG.3E. Tween®20. Detergent concentration: 1% (w/v) lysis temperature: roomtemperature. (solid line A₂₆₀; dotted line A₂₈₀).

[0033]FIG. 4A and FIG. 4B. The HPLC profiles of virus solution before(FIG. 4A) and after (FIG. 4B) Benzonase treatment. (solid line A₂₆₀;dotted line A₂₈₀).

[0034]FIG. 5. The HPLC profile of virus solution after Benzonasetreatment in the presence of 1M NaCl. (solid line A₂₆₀; dotted lineA₂₈₀).

[0035]FIG. 6. Purification of AdCMVp53 virus under buffer A condition of20 mM Tris +1 mM MgCl₂+0.2M NaCl, pH=7.5.

[0036]FIG. 7. Purification of AdCMVp53 virus under buffer A condition of20 mM Tris +1 mM MgCl₂+0.2M NaCl, pH=9.0.

[0037]FIG. 8A, FIG. 8B, and FIG. 8C. HPLC analysis of fractions obtainedduring purification FIG. 8A fraction 3. FIG. 8B fraction 4, FIG. 8Cfraction 8. (solid line A₂₆₀; dotted line A₂₈₀).

[0038]FIG. 9. Purification of AdCMVp53 virus under buffer A condition of20 mM Tris +1mM MgCl₂+0.3M NaCl, pH=9.

[0039]FIG. 10A, FIG. 10B, FIG. 10C, FIG. 10D and FIG. 10E. HPLC analysisof crude virus fractions obtained during purification and CsCl gradientpurified virus. FIG. 10A Crude virus solution. FIG. 10B Flow through.FIG. 10C. Peak number 1. FIG. 10D. Peak number 2. FIG. 10E. CsClpurified virus. (solid line A₂₆₀; dotted line A₂₈₀).

[0040]FIG. 11. HPLC purification profile from a 5 cm id column.

[0041]FIG. 12. The major adenovirus structure proteins detected onSDS-PAGE.

[0042]FIG. 13. The BSA concentration in the purified virus as detectedlevel of the western blot assay. 5 FIG. 14. The chromatogram for thecrude cell lysate material generated from the CellCube™.

[0043]FIG. 15. The elution profile of treated virus solution purifiedusing the method of the present invention using Toyopearl SuperQ resin.

[0044]FIG. 16A and FIG. 16B. HPLC analysis of virus fraction frompurification protocol. FIG. 16A HPLC profiles of virus fraction fromfirst purification step. FIG. 16B HPLC profiles of virus fraction fromsecond purification. (solid line A₂₆₀; dotted line A₂₈₀ ).

[0045]FIG. 17. Purification of 1% Tween® harvest virus solution underlow medium perfusion rate.

[0046]FIG. 18. HPLC analysis of the virus fraction produced under lowmedium perfusion rate.

[0047]FIG. 19A, FIG. 19B and FIG. 19C. Analysis of column purifiedvirus. FIG. 19A SDS-PAGE analysis. FIG. 19B Western blot for BSA. FIG.19C nucleic acid slot blot to determine the contaminating nucleic acidconcentration.

[0048]FIG. 20A, FIG. 20B, FIG. 20C, FIG. 20D, FIG. 20E and FIG. 20F.Capacity study of the Toyopearl SuperQ 650M resin. FIG. 20A Flow throughfrom loading ratio of 1:1. FIG. 20B. Purified virus from loading ratioof 1:1. FIG. 20C Flow through of loading ratio of 2:1. FIG. 20D.Purified virus from the loading ratio of 2:1. FIG. 20E Flow through fromloading ratio of 3:1. FIG. 20F. Purified virus from the loading ratio of3:1. (solid line A₂₆₀; dotted line A₂₈₀).

[0049]FIG. 21. Isopycnic CsCl ultracentrifugation column purified virus.

[0050]FIG. 22. The HPLC profiles of intact viruses present in the columnpurified virus. A. Intact virus B. Defective virus. (solid line A₂₆₀;dotted line A₂₈₀).

[0051]FIG. 23. A production and purification flow chart for AdCMVp53

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0052] It has been shown that adenoviral vectors can successfully beused in eukaryotic gene expression and vaccine development Recently,animal studies have demonstrated that recombinant adenovirus could beused for gene therapy. Successful studies in administering recombinantadenovirus to different tissues have proven the effectiveness ofadenoviral vectors in therapy. This success has led to the use of suchvectors in human clinical trials. There now is an increased demand forthe production of adenoviral vectors to be used in various therapies.The techniques currently available are insufficient to meet such ademand. The present invention provides methods for the production oflarge amounts of adenovirus for use in such therapies.

[0053] The present invention involves a process that has been developedfor the production and purification of a replication deficientrecombinant adenovirus. The production process is based on the use of aCellcube™ bioreactor for cell growth and virus production. It was foundthat a given perfusion rate, used during cell growth and the virusproduction phases of culturing, has a significant effect on thedownstream purification of the virus. More specifically, a low to mediumperfusion rate improves virus production. In addition, lysis solutioncomposed of buffered detergent, used to lyse cells in the Cellcube™ atthe end of virus production phase, also improves the process. With thesetwo advantages, the harvested crude virus solution can be purified usinga single ion exchange chromatography run, afterconcentrationldiafiltration and nuclease treatment to reduce thecontaminating nucleic acid concentration in the crude virus solution.The column purified virus has equivalent purity relative to that ofdouble CsCl gradient purified virus. The total process recovery of thevirus product is 70%±10%. This is a significant improvement over theresults reported by Huyghe et al. (1996). Compared to double CsClgradient ultracentrifugation, column purification has the advantage ofbeing more consistent, scaleable, validatable, faster and lessexpensive. This new process represents a significant improvement in thetechnology for manufacturing of adenoviral vectors for gene therapy.

[0054] Therefore, the present invention is designed to take advantage ofthese improvements in large scale culturing systems and purification forthe purpose of producing and purifying adenoviral vectors. The variouscomponents for such a system, and methods of producing adenovirustherewith, are set forth in detail below.

[0055] 1. Host Cells

[0056] A) Cells

[0057] In a preferred embodiment, the generation and propagation of theadenoviral vectors depend on a unique helper cell line, designated 293,which was transformed from human embryonic kidney cells by Adenovirusserotype 5 (Ad5) DNA fragments and constitutively expresses E1 proteins(Graham et al., 1977). Since the E3 region is dispensable from the Adgenome (Jones and Shenk, 1978), the current Ad vectors, with the help of293 cells, carry foreign DNA in either the E1, the E3 or both regions(Graham and Prevec, 1991; Bett et al., 1994).

[0058] A first aspect of the present invention is the recombinant celllines which express part of the adenoviral genome. These cells lines arecapable of supporting replication of adenovirus recombinant vectors andhelper viruses having defects in certain adenoviral genes, i.e., are“perrnissive” for growth of these viruses and vectors. The recombinant

[0059] cell also is referred to as a helper cell because of the abilityto complement defects in, and support replication of,replication-incompetent adenoviral vectors. The prototype for anadenoviral helper cell is the 293 cell line, which contains theadenoviral E1 region. 293 cells support the replication of adenoviralvectors lacking E1 functions by providing in trans the E1-activeelements necessary for replication.

[0060] Helper cells according to the present invention are derived froma mammalian cell and, preferably, from a primate cell such as humanembryonic kidney cell. Although various primate cells are preferred andhuman or even human embryonic kidney cells are most preferred, any typeof cell that is capable of supporting replication of the virus would beacceptable in the practice of the invention. Other cell types mightinclude, but are not limited to Vero cells, CHO cells or any eukaryoticcells for which tissue culture techniques are established as long as thecells are adenovirus permissive. The term “adenovirus permissive” meansthat the adenovirus or adenoviral vector is able to complete the entireintracellular virus life cycle within the cellular environment.

[0061] The helper cell may be derived from an existing cell line, e.g.,from a 293 cell line, or developed de novo. Such helper cells expressthe adenoviral genes necessary to complement in trans deletions in anadenoviral genome or which supports replication of an otherwisedefective adenoviral vector, such as the E1, E2, E4, E5 and latefunctions.

[0062] A particular portion of the adenovirus genome, the E1 region, hasalready been used to generate complementing cell lines. Whetherintegrated or episomal, portions of the adenovirus genome lacking aviral origin of replication, when introduced into a cell line, will notreplicate even when the cell is superinfected with wild-type adenovirus.In addition, because the transcription of the major late unit is afterviral DNA replication, the late functions of adenovirus cannot beexpressed sufficiently from a cell line. Thus, the E2 regions, whichoverlap with late functions (L1-5), will be provided by helper virusesand not by the cell line. Typically, a cell line according to thepresent invention will express E1 and/or E4.

[0063] As used herein, the term “recombinant” cell is intended to referto a cell into which a gene, such as a gene from the adenoviral genomeor from another cell, has been introduced. Therefore, recombinant cellsare distinguishable from naturally-occurring cells which do not containa recombinantly-introduced gene. Recombinant cells are thus cells havinga gene or genes introduced through “the hand of man.”

[0064] Replication is determined by contacting a layer of uninfectedcells, or cells infected with one or more helper viruses, with virusparticles, followed by incubation of the cells. The formation of viralplaques, or cell free areas in the cell layer, is the result of celllysis caused by the expression of certain viral products. Cell lysis isindicative of viral replication.

[0065] Examples of other useful mammalian cell lines that may be usedwith a replication competent virus or converted into complementing hostcells for use with replication deficient virus are Vero and HeLa cellsand cell lines of Chinese hamster ovary, W138, BHK, COS-7, HepG2, 3T3,RIN and MDCK cells.

[0066] B) Growth in selection media

[0067] In certain embodiments, it may be useful to employ selectionsystems that preclude growth of undesirable cells. This may beaccomplished by virtue of permanently transforming a cell line with aselectable marker or by transducing or infecting a cell line with aviral vector that encodes a selectable marker. In either situation,culture of the transformed/transduced cell with an appropriate drug orselective compound will result in the enhancement, in the cellpopulation, of those cells carrying the marker.

[0068] Examples of markers include, but are not limited to, HSVthymidine kinase, hypoxanthine-guanine phosphoribosyltransferase andadenine phosphoribosyltransferase genes, in tk-, hgprt- or aprt- cells,respectively. Also, anti-metabolite resistance can be used as the basisof selection for dhfr, that confers resistance to methotrexate; gpt,that confers resistance to mycophenolic acid; neo, that confersresistance to the aminoglycoside G418; and hygro, that confersresistance to hygromycin.

[0069] C. Growth in serum weaning

[0070] Serum weaning adaptation of anchorage-dependent cells intoserum-free suspension cultures have been used for the production ofrecombinant proteins (Berg, 1993) and viral vaccines (Perrin, 1995).There have been few reports on the adaptation of 293A cells intoserum-free suspension cultures until recently. Gilbert reported theadaptation of 293A cells into serun-free suspension cultures foradenovirus and recombinant protein production (Gilbert, 1996). Similaradaptation method had been used for the adaptation of A549 cells intoserum-free suspension culture for adenovirus production (Morris et al.,1996). Cell-specific virus yields in the adapted suspension cells,however, are about 5-10-fold lower than those achieved in the parentalattached cells.

[0071] Using the similar serum weaning procedure, the inventors havesuccessfully adapted the 293A cells into serum-free suspension culture(293SF cells). In this procedure, the 293 cells were adapted to acommercially available 293 media by sequentially lowering down the FBSconcentration in T-flasks. Briefly, the initial serum concentration inthe media was approximately 10% FBS DMEM media in T-75 flask and thecells were adapted to serum-free IS 293 media in T-flasks by loweringdown the FBS concentration in the media sequentially. After 6 passagesin T-75 flasks the FBS% was estimated to be about 0.019% and the 293cells. The cells were subcultured two more times in the T flasks beforethey were transferred to spinner flasks. The results described hereinbelow show that cells grow satisfactorily in the serum-free medium(IS293 medium, Irvine Scientific, Santa Ana, Calif.). Average doublingtime of the cells were 18-24 h achieving stationary cell concentrationsin the order of 4-10×106 cells/ml without medium exchange.

[0072] D. Adaptation of cellsfor Suspension Culture

[0073] Two methodologies have been used to adapt 293 cells intosuspension cultures. Graham adapted 293A cells into suspension culture(293N3S cells) by 3 serial passages in nude mice (Graham, 1987). Thesuspension 293N3S cells were found to be capable of supporting E1adenoviral vectors. However, Gamier et al. (1994) observed that the293N35 cells had a relatively long initial. lag phase in suspension, alow growth rate, and a strong tendency to clump.

[0074] The second method that has been used is a gradual adaptation of293A cells into suspension growth (Cold Spring Harbor Laboratories, 293Scells). Garnier et al. (1994) reported the use of 293S cells forproduction of recombinant proteins from adenoviral vectors. The authorsfound that 293S cells were much less clumpy in calcium-free media and afresh medium exchange at the time of virus infection could significantlyincrease the protein production. It was found that glucose was thelimiting factor in culture without medium exchange.

[0075] In the present invention, the 293 cells adapted for growth inserun-free conditions were adapted into a suspension culture. The cellswere transferred in a serun-free 250 mL spinner suspension culture (100mL working volume) for the suspension culture at an initial cell densityof between about 1.18E+5 vc/mL and about 5.22E+5 vc/mL. The media may besupplemented with heparin to prevent aggregation of cells. This cellculture systems allows for some increase of cell density whilst cellviability is maintained. Once these cells are growing in culture, theycells are subcultured in the spinner flasks approximately 7 morepassages. It may be noted that the doubling time of the cells isprogressively reduced until at the end of the successive passages thedoubling time is about 1.3 day, ie. comparable to 1.2 day of the cellsin 10% FBS media in the attached cell culture. In the serum-free IS 293media supplemented with heparin almost all the cells existed asindividual cells not forming aggregates of cells in the suspensionculture.

[0076] 2. Cell Culture Systems

[0077] The ability to produce infectious viral vectors is increasinglyimportant to the pharmaceutical industry, especially in the context ofgene therapy. Over the last decade, advances in biotechnology have ledto the production of a number of important viral vectors that havepotential uses as therapies, vaccines and protein production machines.The use of viral vectors in mamnalian cultures has advantages overproteins produced in bacterial or other lower lifeform hosts in theirability to post-translationally process complex protein structures suchas disulfide-dependent folding and glycosylation.

[0078] Development of cell culture for production of virus vectors hasbeen greatly aided by the development in molecular biology of techniquesfor design and construction of vector systems highly efficient inmammalian cell cultures, a battery of useful selection markers, geneamplification schemes and a more comprehensive understanding of thebiochemical and cellular mechanisms involved in procuring the finalbiologically-active molecule from the introduced vector.

[0079] Frequently, factors which affect the downstream (in this case,beyond the cell lysis) side of manufacturing scale-up were notconsidered before selecting the cell line as the host for the expressionsystem. Also, development of bioreactor systems capable of sustainingvery high density cultures for prolonged periods of time have not livedup to the increasing demand for increased production at lower costs.

[0080] The present invention will take advantage of the recentlyavailable bioreactor technology. Growing cells according to the presentinvention in a bioreactor allows for large scale production of fullybiologically-active cells capable of being infected by the adenoviralvectors of the present invention. By operating the system at a lowperfusion rate and applying a different scheme for purification of theinfecting particles, the invention provides a purification strategy thatis easily scaleable to produce large quantities of highly purifiedproduct.

[0081] Bioreactors have been widely used for the production ofbiological products from both suspension and anchorage dependent animalcell cultures. The most widely used producer cells for adenoviral vectorproduction are anchorage dependent human embryonic kidney cells (293cells). Bioreactors to be developed for adenoviral vector productionshould have the characteristic of high volume-specific culture surfacearea in order to achieve high producer cell density and high virusyield. Microcarrier cell culture in stirred tank bioreactor providesvery high volume-specific culture surface area and has been used for theproduction of viral vaccines (Griffiths, 1986). Furthermore, stirredtank bioreactors have industrially been proven to be scaleable. Themultiplate Cellcube™ cell culture system manufactured by Corning-Costaralso offers a very high volume-specific culture surface area. Cells growon both sides of the culture plates hermetically sealed together in theshape of a compact cube. Unlike stirred tank bioreactors, the Cellcube™culture unit is disposable. This is very desirable at the early stageproduction of clinical product because of the reduced capitalexpenditure, quality control and quality assurance costs associated withdisposable systems. In consideration of the advantages offered by thedifferent systems, both the stirred tank bioreactor and the Cellcube™system were evaluated for the production of adenovirus.

[0082] A) Anchorage-dependent versus non-anchorage-dependent cultures.

[0083] Animal and human cells can be propagated in vitro in two modes:as non-anchorage dependent cells growing freely in suspension throughoutthe bulk of the culture; or as anchorage-dependent cells requiringattachment to a solid substrate for their propagation (ie., a monolayertype of cell growth).

[0084] Non-anchorage dependent or suspension cultures from continuousestablished cell lines are the most widely used means of large scaleproduction of cells and cell products. Large scale suspension culturebased on microbial (bacterial and yeast) fermentation technology hasclear advantages for the manufacturing of mammalian cell products. Theprocesses are relatively simple to operate and straightforward to scaleup. Homogeneous conditions can be provided in the reactor which allowsfor precise monitoring and control of temperature, dissolved oxygen, andpH, and ensure that representative samples of the culture can be taken.

[0085] However, suspension cultured cells cannot always be used in theproduction of biologicals. Suspension cultures are still considered tohave tumorigenic potential and thus their use as substrates forproduction put limits on the use of the resulting products in human andveterinary applications (Petricciani, 1985; Larsson, 1987). Virusespropagated in suspension cultures as opposed to anchorage-dependentcultures can sometimes cause rapid changes in viral markers, leading toreduced immunogenicity (Bahnemann, 1980). Finally, sometimes evenrecombinant cell lines can secrete considerably higher amounts ofproducts when propagated as anchorage-dependent cultures as comparedwith the same cell line in suspension (Nilsson and Mosbach, 1987). Forthese reasons, different types of anchorage-dependent cells are usedextensively in the production of different biological products.

[0086] B) Reactors and processes for suspension.

[0087] Large scale suspension culture of mammalian cultures in stirredtanks was undertaken. The instrumentation and controls for bioreactorsadapted, along with the design of the fermentors, from related microbialapplications. However, acknowledging the increased demand forcontamination control in the slower growing mammalian cultures, improvedaseptic designs were quickly implemented, improving dependability ofthese reactors. Instrumentation and controls are basically the same asfound in other fermentors and include agitation, temperature, dissolvedoxygen, and pH controls. More advanced probes and autoanalyzers foron-line and off-line measurements of turbidity (a function of particlespresent), capacitance (a function of viable cells present),glucose/lactate, carbonate/bicarbonate and carbon dioxide are available.Maximum cell densities obtainable in suspension cultures are relativelylow at about 2-4×106 cellslrnl of medium (which is less than 1 mg drycell weight per ml), well below the numbers achieved in microbialfermentation.

[0088] Two suspension culture reactor designs are most widely used inthe industry due to their simplicity and robustness of operation—thestirred reactor and the airlift reactor. The stirred reactor design hassuccessfully been used on a scale of 8000 liter capacity for theproduction of interferon (Phillips et al., 1985; Mizrahi, 1983). Cellsare grown in a stainless steel tank with a height-to-diameter ratio of1:1 to 3:1. The culture is usually mixed with one or more agitators,based on bladed disks or marine propeller patterns. Agitator systemsoffering less shear forces than blades have been described. Agitationmay be driven either directly or indirectly by magnetically coupleddrives. Indirect drives reduce the risk of microbial contaminationthrough seals on stirrer shafts.

[0089] The airlift reactor, also initially described for microbialfermentation and later adapted for mammalian culture, relies on a gasstream to both mix and oxygenate the culture. The gas stream enters ariser section of the reactor and drives circulation. Gas disengages atthe culture surface, causing denser liquid free of gas bubbles to traveldownward in the downcomer section of the reactor. The main advantage ofthis design is the simplicity and lack of need for mechanical mixing.Typically, the height-to-diameter ratio is 10:1. The airlift reactorscales up relatively easily, has good mass transfer of gasses andgenerates relatively low shear forces.

[0090] Most large-scale suspension cultures are operated as batch orfed-batch processes because they are the most straightforward to operateand scale up. However, continuous processes based on chemostat orperfusion principles are available.

[0091] A batch process is a closed system in which typical growthprofile is seen. A lag phase is followed by exponential, stationary anddecline phases. In such a system, the environment is continuouslychanging as nutrients are depleted and metabolites accumulate. Thismakes analysis of factors influencing cell growth and productivity, andhence optimization of the process, a complex task. Productivity of abatch process may be increased by controlled feeding of key nutrients toprolong the growth cycle. Such a fed-batch process is still a closedsystem because cells, products and waste products are not removed.

[0092] In what is still a closed system, perfusion of fresh mediumthrough the culture can be achieved by retaining the cells with avariety of devices (e.g. fine mesh spin filter, hollow fiber or flatplate membrane filters, settling tubes). Spin filter cultures canproduce cell densities of approximately 5×107 cells/ml. A true opensystem and the simplest perfusion process is the chemostat in whichthere is an inflow of medium and an outflow of cells and products.Culture medium is fed to the reactor at a predetermined and constantrate which maintains the dilution rate of the culture at a value lessthan the maximum specific growth rate of the cells (to prevent washoutof the cell mass from the reactor). Culture fluid containing cells andcell products and byproducts is removed at the same rate.

[0093] C) Non-perfused attachment systems.

[0094] Traditionally, anchorage-dependent cell cultures are propagatedon the bottom of small glass or plastic vessels. The restrictedsurface-to-volume ratio offered by classical and traditional techniques,suitable for the laboratory scale, has created a bottleneck in theproduction of cells and cell products on a large scale. In an attempt toprovide systems that offer large accessible surfaces for cell growth insmall culture volume, a number of techniques have been proposed: theroller bottle system, the stack plates propagator, the spiral filmbottles, the hollow fiber system, the packed bed, the plate exchangersystem, and the membrane tubing reel. Since these systems arenon-homogeneous in their nature, and are sometimes based on multipleprocesses, they suffer from the following shortcomings—limited potentialfor scale-up, difficulties in taking cell samples, limited potential formeasuring and controlling key process parameters and difficulty inmaintaining homogeneous environmental conditions throughout the culture.

[0095] Despite these drawbacks, a commonly used process for large scaleanchorage-dependent cell production is the roller bottle. Being littlemore than a large, differently shaped T-flask, simplicity of the systemmakes it very dependable and, hence, attractive. Fully automated robotsare available that can handle thousands of roller bottles per day, thuseliminating the risk of contamination and inconsistency associated withthe otherwise required intense human handling. With frequent mediachanges, roller bottle cultures can achieve cell densities of close to0.5×106 cells/cm² (corresponding to approximately 10⁹ cellsfbottle oralmost 10⁷ cells/ml of culture media).

[0096] D) Cultures on microcarriers

[0097] In an effort to overcome the shortcomings of the traditionalanchorage-dependent culture processes, van Wezel (1967) developed theconcept of the microcarrier culturing systems. In this system, cells arepropagated on the surface of small solid particles suspended in thegrowth medium by slow agitation. Cells attach to the microcarriers andgrow gradually to confluency on the microcarrier surface. In fact, thislarge scale culture system upgrades the attachment dependent culturefrom a single disc process to a unit process in which both monolayer andsuspension culture have been brought together. Thus, combining thenecessary surface for a cell to grow with the advantages of thehomogeneous suspension culture increases production.

[0098] The advantages of microcarrier cultures over most otheranchorage-dependent, large-scale cultivation methods are several fold.First, microcarrier cultures offer a high surface-to-volume ratio(variable by changing the carrier concentration) which leads to highcell density yields and a potential for obtaining highly concentratedcell products. Cell yields are up to 1-2×10⁷ cells/ml when cultures arepropagated in a perfused reactor mode. Second, cells can be propagatedin one unit process vessels instead of using many small low-productivityvessels (i.e., flasks or dishes). This results in far better nutrientutilization and a considerable saving of culture medium. Moreover,propagation in a single reactor leads to reduction in need for facilityspace and in the number of handling steps required per cell, thusreducing labor cost and risk of contamination. Third, the well-mixed andhomogeneous microcarrier suspension culture makes it possible to monitorand control environmental conditions (e.g., pH, PO₂, and concentrationof medium components), thus leading to more reproducible cellpropagation and product recovery. Fourth, it is possible to take arepresentative sample for microscopic observation, chemical testing, orenumeration. Fifth, since microcarriers settle out of suspensionquickly, use of a fed-batch process or harvesting of cells can be donerelatively easily. Sixth, the mode of the anchorage-dependent culturepropagation on the microcarriers makes it possible to use this systemfor other cellular manipulations, such as cell transfer without the useof proteolytic enzymes, cocultivation of cells, transplantation intoanimals, and perfusion of the culture using decanters, columns,fluidized beds, or hollow fibers for microcarrier retainment. Seventh,microcarrier cultures are relatively easily scaled up using conventionalequipment used for cultivation of microbial and animal cells insuspension.

[0099] E) Microencapsulation of mammalian cells

[0100] ne method which has shown to be particularly useful for culturingmammalian cells is microencapsulation. The mammalian cells are retainedinside a semipermeable hydrogel membrane. A porous membrane is formedaround the cells permitting the exchange of nutrients, gases, andmetabolic products with the bulk medium surrounding the capsule. Severalmethods have been developed that are gentle, rapid and non-toxic andwhere the resulting membrane is sufficiently porous and strong tosustain the growing cell mass throughout the term of the culture. Thesemethods are all based on soluble alginate gelled by droplet contact witha calcium-containing solution. Lim (1982, U.S. Pat. No. 4,352,883,incorporated herein by reference,) describes cells concentrated in anapproximately 1% solution of sodium alginate which are forced through asmall orifice, forming droplets, and breaking free into an approximately1% calcium chloride solution. The droplets are then cast in a layer ofpolyamino acid that ionically bonds to the surface alginate. Finally thealginate is reliquefied by treating the droplet in a chelating agent toremove the calcium ions. Other methods use cells in a calcium solutionto be dropped into a alginate solution, thus creating a hollow alginatesphere. A similar approach involves cells in a chitosan solution droppedinto alginate, also creating hollow spheres.

[0101] Microencapsulated cells are easily propagated in stirred tankreactors and, with beads sizes in the range of 150-1500 μm in diameter,are easily retained in a perfused reactor using a fine-meshed screen.The ratio of capsule volume to total media volume can be maintained fromas dense as 1:2 to 1:10. With intracapsular cell densities of up to 10⁸,the effective cell density in the culture is 1-5×10⁷.

[0102] The advantages of microencapsulation over other processes includethe protection from the deleterious effects of shear stresses whichoccur from sparging and agitation, the ability to easily retain beadsfor the purpose of using perfused systems, scale up is relativelystraightforward and the ability to use the beads for implantation.

[0103] The current invention includes cells which areanchorage-dependent in nature. 293 cells, for example, areanchorage-dependent, and when grown in suspension, the cells will attachto each other and grow in clumps, eventually suffocating cells in theinner core of each clump as they reach a size that leaves the core cellsunsustainable by the culture conditions. Therefore, an efficient meansof large-scale culture of anchorage-dependent cells is needed in orderto effectively employ these cells to generate large quantities ofadenovirus.

[0104] F) Perfused attachment systems

[0105] Perfused attachment systems are a preferred form of the presentinvention. Perfusion refers to continuous flow at a steady rate, throughor over a population of cells (of a physiological nutrient solution). Itimplies the retention of the cells within the culture unit as opposed tocontinuous-flow culture which washes the cells out with the withdrawnmedia (e g., chemostat). The idea of perfusion has been known since thebeginning of the century, and has been applied to keep small pieces oftissue viable for extended microscopic observation. The technique wasinitiated to mimic the cells milieu in vivo where cells are continuouslysupplied with blood, lymph, or other body fluids. Without perfusion,cells in culture go through alternating phases of being fed and starved,thus limiting full expression of their growth and metabolic potential.

[0106] The current use of perfused culture is in response to thechallenge of growing cells at high densities (i.e., 0.1-5×10⁸ cells/ml).In order to increase densities beyond 2-4×106 cells/ml, the medium hasto be constantly replaced with a fresh supply in order to make up fornutritional deficiencies and to remove toxic products. Perfusion allowsfor a far better control of the culture environment (pH, PO₂, nutrientlevels, etc.) and is a means of significantly increasing the utilizationof the surface area within a culture for cell attachment.

[0107] The development of a perfused packed-bed reactor using a bedmatrix of a non-woven fabric has provided a means for maintaining aperfusion culture at densities exceeding 108 cells/ml of the bed volume(CelliGen™, New Brunswick Scientific, Edison, N.J.; Wang et al., 1992;Wang et al., 1993; Wang et al., 1994). Briefly described, this reactorcomprises an improved reactor for culturing of both anchorage- andnon-anchorage-dependent cells. The reactor is designed as a packed bedwith a means to provide internal recirculation. Preferably, a fibermatrix carrier is placed in a basket within the reactor vessel. A topand bottom portion of the basket has holes, allowing the medium to flowthrough the basket. A specially designed impeller provides recirculationof the medium through the space occupied by the fiber matrix forassuring a uniform supply of nutrient and the removal of wastes. Thissimultaneously assures that a negligible amount of the total cell massis suspended in the medium. The combination of the basket and therecirculation also provides a bubble-free flow of oxygenated mediumthrough the fiber matrix. The fiber matrix is a non-woven fabric havinga “pore” diameter of from 10 μm to 100 μm, providing for a high internalvolume with pore volumes corresponding to 1 to 20 times the volumes ofindividual cells.

[0108] In comparison to other culturing systems, this approach offersseveral significant advantages. With a fiber matrix carrier, the cellsare protected against mechanical stress from agitation and foaming. Thefree medium flow through the basket provides the cells with optimumregulated levels of oxygen, pH, and nutrients. Products can becontinuously removed from the culture and the harvested products arefree of cells and can be produced in low-protein medium whichfacilitates subsequent purification steps. Also, the unique design ofthis reactor system offers an easier way to scale up the reactor.Currently, sizes up to 30 liter are available. One hundred liter and 300liter versions are in development and theoretical calculations supportup to a 1000 liter reactor. This technology is explained in detail in WO94/17178 (Aug. 4, 1994, Freedman et al.), which is hereby incorporatedby reference in its entirety.

[0109] The Cellcube™ Coming-Costar) module provides a large styrenicsurface area for the immobilization and growth of substrate attachedcells. It is an integrally encapsulated sterile single-use device thathas a series of parallel culture plate joined to create thin sealedlaminar flow spaces between adjacent plates.

[0110] The Cellcube™ module has inlet and outlet ports that arediagonally opposite each other and help regulate the flow of media.During the first few days of growth the culture is generally satisfiedby the media contained within the system after initial seeding. Theamount of time between the initial seeding and the start of the mediaperfusion is dependent on the density of cells in the seeding inoculumand the cell growth rate. The measurement of nutrient concentration inthe circulating media is a good indicator of the status of the culture.When establishing a procedure it may be necessary to monitor thenutrients composition at a variety of different perfusion rates todetermine the most economical and productive operating parameters.

[0111] Cells within the system reach a higher density of solution(cells/ml) than in traditional culture systems. Many typically usedbasal media are designed to support 1-2×10⁶ cells/ml/day. A typicalCellcube™, run with an 85,000 cm² surface, contains approximately 6Lmedia within the module. The cell density often exceeds 10⁷ cells/mL inthe culture vessel. At confluence, 2-4 reactor volumes of media arerequired per day.

[0112] The timing and parameters of the production phase of culturesdepends on the type and use of a particular cell line. Many culturesrequire a different media for production than is required for the growthphase of the culture. The transition from one phase to the other willlikely require multiple washing steps in traditional cultures. However,the Cellcube™ system employs a perfusion system. On of the benefits ofsuch a system is the ability to provide a gentle transition betweenvarious operating phases. The perfusion system negates the need fortraditional wash steps that seek to remove serum components in a growthmedium.

[0113] In an exemplary embodiment of the present invention, theCellCube™ system is used to grow cells transfected with AdCMVp53. 293cells were inoculated into the Cellcube™ according to the manufacturer'srecommendation. Inoculation cell densities were in the range of1-1.5×10⁴/cm². Cells were allowed to grow for 7 days at 37° C. underculture conditions of pH=7.20, DO=60% air saturation. The mediumperfusion rate was regulated according to the glucose concentration inthe Cellcube™. One day before viral infection, medium for perfusion waschanged from a buffer comprising 10% FBS to a buffer comprising 2% FBS.On day 8, cells were infected with virus at a multiplicity of infection(MOI) of 5. Medium perfusion was stopped for 1 hr immediately afterinfection then resumed for the remaining period of the virus productionphase. Culture was harvested 45-48 hr post-infection. Of course theseculture conditions are exemplary and may be varied according to thenutritional needs and growth requirements of a particular cell line.Such variation may be performed without undue experimentation and arewell within the skill of the ordinary person in the art.

[0114] G) Serum-Free Suspension Culture

[0115] In particular embodiments, adenoviral vectors for gene therapyare produced from anchorage-ependent culture of 293 cells (293A cells)as described above. Scale-up of adenoviral vector production isconstrained by the anchorage-dependency of 293A cells. To facilitatescale-up and meet future demand for adenoviral vectors, significantefforts have been devoted to the development of alternative productionprocesses that are amenable to scale-up. Methods include growing 293Acells in microcarrier cultures and adaptation of 293A producer cellsinto suspension cultures. Microcarrier culture techniques have beendescribed above. This technique relies on the attachment of producercells onto the surfaces of microcarriers which are suspended in culturemedia by mechanical agitation. The requirement of cell attachment maypresent some limitations to the scaleability of microcarrier cultures.

[0116] Until the present application there have been no reports on theuse of 293 suspension cells for adenoviral vector production for genetherapy. Furthermore, the reported suspension 293 cells require thepresence of 5-10% FBS in the culture media for optimal cell growth andvirus production. Historically, presence of bovine source proteins incell culture media has been a regulatory concerns, especially recentlybecause of the outbreak of Bovine Spongiform Encephalopathy (BSE) insome countries. Rigorous and complex downstream purification process hasto be developed to remove contaminating proteins and any adventitiousviruses from the final product. Development of serum-free 293 suspensionculture is deemed to be a major process improvement for the productionof adenoviral vector for gene therapy.

[0117] Results of virus production in spinner flasks and a 3 L stirredtank bioreactor indicate that cell specific virus productivity of the293SF cells was approximately 2.5×10⁴ vp/cell, which is approximately60-90% of that from the 293A cells. However, because of the higherstationary cell concentration, volumetric virus productivity from the293SF culture is essentially equivalent to that of the 293A cellculture. The inventors also observed that virus production increasedsignificantly by carrying out a fresh medium exchange at the time ofvirus infection. The inventors are going to evaluate the limitingfactors in the medium.

[0118] These findings allow for a scaleable, efficient, and easilyvalidatable process for the production adenoviral vector. Thisadaptation method is not limited to 293A cells only and will be equallyusefull when applied to other adenoviral vector producer cells.

[0119] 3. Methods of Cell Harvest and Lysis

[0120] Adenoviral infection results in the lysis of the cells beinginfected. The lytic characteristics of adenovirus infection permit twodifferent modes of virus production. One is harvesting infected cellsprior to cell lysis. The other mode is harvesting virus supernatantafter complete cell lysis by the produced virus. For the latter mode,longer incubation times are required in order to achieve complete celllysis. This prolonged incubation time after virus infection creates aserious concern about increased possibility of generation of replicationcompetent adenovirus (RCA), particularly for the current firstgeneration adenoviral vectors (E1-deleted vector). Therefore, harvestinginfected cells before cell lysis was chosen as the production mode ofchoice. Table 1 lists the most common methods that have been used forlysing cells after cell harvest. TABLE 1 Methods used for cell lysisMethods Procedures Comments Freeze-thaw Cycling between Easy to carrydry ice out at lab and 37° C. scale. High cell water bath lysisefficiency Not scaleable Not recommended for large scale manufacturingSolid Shear French Press Capital equipment Hughes Press investment Viruscontainment concerns Lack of experience Detergent Non-ionic Easy tocarry out lysis detergent at both lab solutions such and manufacturingas Tween, scale Triton, NP-40, etc. Wide variety of detergent choicesConcerns of residual detergent in finished product Hypotonic water,citric Low lysis solution lysis buffer efficiency Liquid ShearHomogenizer Capital equipment Impinging Jet investment MicrofluidizerVirus containment concerns Scaleability concerns Sonication ultrasoundCapital equipment investment Virus containment concerns Noise pollutionScaleability concern

[0121] A) Detergents

[0122] Cells are bounded by membranes. In order to release components ofthe cell, it is necessary to break open the cells. The most advantageousway in which this can be accomplished, according to the presentinvention, is to solubilize the membranes with the use of detergents.Detergents are amphipathic molecules with an apolar end of aliphatic oraromatic nature and a polar end which may be charged or uncharged.Detergents are more hydrophilic than lipids and thus have greater watersolubility than lipids. They allow for the dispersion of water insolublecompounds into aqueous media and are used to isolate and purify proteinsin a native form.

[0123] Detergents can be denaturing or non-denaturing. The former can beanionic such as sodium dodecyl sulfate or cationic such as ethyltrimethyl ammonium bromide. These detergents totally disrupt membranesand denature the protein by breaking protein-protein interactions. Nondenaturing detergents can be divided into non-anionic detergents such asTriton®X-100, bile salts such as cholates and zwitterionic detergentssuch as CHAPS. Zwitterionics contain both cationic and anion groups inthe same molecule, the positive electric charge is neutralized by thenegative charge on the same or adjacent molecule.

[0124] Denaturing agents such as SDS bind to proteins as monomers andthe reaction is equilibrium driven until saturated. Thus, the freeconcentration of monomers determines the necessary detergentconcentration. SDS binding is cooperative i.e. the binding of onemolecule of SDS increase the probability of another molecule binding tothat protein, and alters proteins into rods whose length is proportionalto their molecular weight.

[0125] Non-denaturing agents such as Triton®X-100 do not bind to nativeconformations nor do they have a cooperative binding mechanism. Thesedetergents have rigid and bulky apolar moieties that do not penetrateinto water soluble proteins. They bind to the hydrophobic parts ofproteins. Triton®X100 and other polyoxyethylene nonanionic detergentsare inefficient in breaking protein-protein interaction and can causeartifactual aggregations of protein. These detergents will, however,disrupt protein-lipid interactions but are much gentler and capable ofmaintaining the native form and functional capabilities of the proteins.

[0126] Detergent removal can be attempted in a number of ways. Dialysisworks well with detergents that exist as monomers. Dialysis is somewhatineffective with detergents that readily aggregate to form micellesbecause they micelles are too large to pass through dialysis. Ionexchange chromatography can be utilized to circumvent this problem. Thedisrupted protein solution is applied to an ion exchange chromatographycolumn and the column is then washed with buffer minus detergent. Thedetergent will be removed as a result of the equilibration of the bufferwith the detergent solution. Alternatively the protein solution may bepassed through a density gradient. As the protein sediments through thegradients the detergent will come off due to the chemical potential.

[0127] Often a single detergent is not versatile enough for thesolubilization and analysis of the milieu of proteins found in a cell.The proteins can be solubilized in one detergent and then placed inanother suitable detergent for protein analysis. The protein detergentmicelles formed in the first step should separate from pure detergentmicelles. When these are added to an excess of the detergent foranalysis, the protein is found in micelles with both detergents.Separation of the detergent-protein micelles can be accomplished withion exchange or gel filtration chromatography, dialysis or buoyantdensity type separations.

[0128] Triton®X- Detergents: This family of detergents (Triton®100, X114and NP-40) have the same basic characteristics but are different intheir specific hydrophobic-hydrophilic nature. All of theseheterogeneous detergents have a branched 8-carbon chain attached to anaromatic ring. This portion of the molecule contributes most of thehydrophobic nature of the detergent. Triton®X detergents are used tosolublize membrane proteins under non-denaturing conditions. The choiceof detergent to solubilize proteins will depend on the hydrophobicnature of the protein to be solubilized. Hydrophobic proteins requirehydrophobic detergents to effectively solubilize them.

[0129] Triton®X-100 and NP-40 are very similar in structure andhydrophobicity and are interchangeable in most applications includingcell lysis, delipidation protein dissociation and membrane protein andlipid solubilization. Generally 2 mg detergent is used to solubilize lmgmembrane protein or 10 mg detergent/1 mg of lipid membrane. Triton®X-114is useful for separating hydrophobic frqm hydrophilic proteins.

[0130] Brij®X Detergents: These are similar in structure to Triton®Xdetergents in that they have varying lengths of polyoxyethylene chainsattached to a hydrophobic chain. However, unlike Triton®X detergents,the Brij® detergents do not have an aromatic ring and the length of thecarbon chains can vary. The Brij detergents are difficult to remove fromsolution using dialysis but may be removed by detergent removing gels.Brij®58 is most similar to Triton®X100 in its hydrophobic/hydrophiliccharacteristics. Brij®-35 is a commonly used detergent in HPLCapplications.

[0131] Dializable Nonionic Detergents: η-Octyl-β-D-glucoside(octylglucopyranoside) and η-Octyl-β-D-thioglucoside(octylthioglucopyranoside, OTG) are nondenaturing nonionic detergentswhich are easily dialyzed from solution. These detergents are useful forsolubilizing membrane proteins and have low UV absorbances at 280 nm.Octylglucoside has a high CMC of 23-25 mM and has been used atconcentrations of 1.1-1.2% to solubilize membrane proteins.

[0132] Octylthioglucoside was first synthesized to offer an alternativeto octylglucoside. Octylglucoside is expensive to manufacture and thereare some inherent problems in biological systems because it can behydrolyzed by β-glucosidase.

[0133] Tween® Detergents: The Tween® detergents are nondenaturing,nonionic detergents. They are polyoxyethylene sorbitan esters of fattyacids. Tween® 20 and Tween® 80 detergents are used as blocking agents inbiochemical applications and are usually added to protein solutions toprevent nonspecific binding to hydrophobic materials such as plastics ornitrocellulose. They have been used as blocking agents in ELISA andblotting applications. Generally, these detergents are used atconcentrations of 0.01-1.0% to prevent nonspecific binding tohydrophobic materials.

[0134] Tween® 20 and other nonionic detergents have been shown to removesome proteins from the surface of nitrocellulose. Tween® 80 has beenused to solubilize membrane proteins, present nonspecific binding ofprotein to multiwell plastic tissue culture plates and to reducenonspecific binding by serum proteins and biotinylated protein A topolystyrene plates in ELISA.

[0135] The difference between these detergents is the length of thefatty acid chain. Tween® 80 is derived from oleic acid with a C₁₈ chainwhile Tween® 20 is derived from lauric acid with a C₁₂ chain. The longerfatty acid chain makes the Tween® 80 detergent less hydrophilic thanTween® 20 detergent. Both detergents are very soluble in water.

[0136] The Tween® detergents are difficult to remove from solution bydialysis, but Tween® 20 can be removed by detergent removing gels. Thepolyoxyethylene chain found in these detergents makes them subject tooxidation (peroxide formation) as is true with the Triton®X and Brij®series detergents.

[0137] Zwitterionic Detergents: The zwitterionic detergent, CHAPS, is asulfobetaine derivative of cholic acid. This zwitterionic detergent isuseful for membrane protein solubilization when protein activity isimportant. This detergent is useful over a wide range of pH (pH 2-12)and is easily removed from solution by dialysis due to high CMCs (8-10mM). This detergent has low absorbances at 280 nm making it useful whenprotein monitoring at this wavelength is necessary. CHAPS is compatiblewith the BCA Protein Assay and can be removed from solution by detergentremoving gel. Proteins can be iodinated in the presence of CHAPS

[0138] CHAPS has been successfully used to solubilize intrinsic membraneproteins and receptors and maintain the functional capability of theprotein. When cytochrome P450 is solubilized in either Triton®X-100 orsodium cholate aggregates are formed.

[0139] B) Non-Detergent Methods

[0140] Various non-detergent methods, though not preferred, may beemployed in conjunction with other advantageous aspects of the presentinvention:

[0141] Freeze-Thaw: This has been a widely used technique for lysiscells in a gentle and effective manner. Cells are generally frozenrapidly in, for example, a dry ice/ethanol bath until completely frozen,then transferred to a 37° C. bath until completely thawed. This cycle isrepeated a number of times to achieve complete cell lysis.

[0142] Sonication: High frequency ultrasonic oscillations have beenfound to be useful for cell disruption. The method by which ultrasonicwaves break cells is not fully understood but it is known that hightransient pressures are produced when suspensions are subjected toultrasonic vibration. The main disadvantage with this technique is thatconsiderable amounts of heat are generated. In order to minimize heateffects specifically designed glass vessels are used to hold the cellsuspension. Such designs allow the suspension to circulate away from theultrasonic probe to the outside of the vessel where it is cooled as theflask is suspended in ice.

[0143] High Pressure Extrusion: This is a frequently used method todisrupt microbial cell. The French pressure cell employs pressures of10.4×10⁷ Pa (16, 000 p.s.i) to break cells open. These apparatusconsists of a stainless steel chamber which opens to the outside bymeans of a needle valve. The cell suspension is placed in the chamberwith the needle valve in the closed position. After inverting thechamber, the valve is opened and the piston pushed in to force out anyair in the chamber. With the valve in the closed position, the chamberis restored to its original position, placed on a solid based and therequired pressure is exerted on the piston by a hydraulic press. Whenthe pressure has been attained the needle valve is opened fractionallyto slightly release the pressure and as the cells expand they burst. Thevalve is kept open while the pressure is maintained so that there is atrickle of ruptured cell which may be collected.

[0144] Solid Shear Methods: Mechanical shearing with abrasives may beachieved in Mickle shakers which oscillate suspension vigorously(300-3000 time/min) in the presence of glass beads of 500 nm diameter.This method may result in organelle damage. A more controlled method isto use a Hughes press where a piston forces most cells together withabrasives or deep frozen paste of cells through a 0.25 mm diameter slotin the pressure chamber. Pressures of up to 5.5×10⁷ Pa (8000 p.s.i.) maybe used to lyse bacterial preparations.

[0145] Liquid Shear Methods: These methods employ blenders, which usehigh speed reciprocating or rotating blades, homogenizers which use anupward/downward motion of a plunger and ball and microfluidizers orimpinging jets which use high velocity passage through small diametertubes or high velocity impingement of two fluid streams. The blades ofblenders are inclined at different angles to permit efficient mixing.Homogenizers are usually operated in short high speed bursts of a fewseconds to minimize local heat. These techniques are not generallysuitable for microbial cells but even very gentle liquid shear isusually adequate to disrupt animal cells.

[0146] Hypotonic/Hypertonic Methods: Cells are exposed to a solutionwith a much lower (hypotonic) or higher (hypertonic) soluteconcentration. The difference in solute concentration creates an osmoticpressure gradient. The resulting flow of water into the cell in ahypotonic environment causes the cells to swell and burst The flow ofwater out of the cell in a hypertonic environment causes the cells toshrink and subsequently burst.

[0147] 4. Methods of Concentration and Filtration

[0148] One aspect of the present invention employs methods of crudepurification of adenovirus from a cell lysate. These methods includeclarification, concentration and diafiltration. The initial step in thispurification process is clarification of the cell lysate to remove largeparticulate matter, particularly cellular components, from the celllysate. Clarification of the lysate can be achieved using a depth filteror by tangential flow filtration. In a preferred embodiment of thepresent invention, the cell lysate is passed through a depth filter,which consists of a packed column of relatively non-adsorbent material(e.g. polyester resins, sand, diatomeceous earth, colloids, gels, andthe like). In tangential flow filtration (TFF), the lysate solutionflows across a membrane surface which facilitates back diffusion ofsolute from the membrane surface into the bulk solution. Membranes aregenerally arranged within various types of filter apparatus includingopen channel plate and frame, hollow fibers, and tubules.

[0149] After clarification and prefiltration of the cell lysate, theresultant virus supernatant is first concentrated and then the buffer isexchanged by diafiltration. The virus supernatant is concentrated bytangential flow filtration across an ultrafiltration membrane of100-300K nominal molecular weight cutoff. Ultrafiltration is apressure-modified convective process that uses semi-perneable membranesto separate species by molecular size, shape and/or charge. It separatessolvents from solutes of various sizes, independent of solute molecularsize. Ultrafiltration is gentle, efficient and can be used tosimultaneously concentrate and desalt solutions. Ultrafiltrationmembranes generally have two distinct layers: a thin (0.1-1.5 μm), denseskin with a pore diameter of 10400 angstroms and an open substructure ofprogressively larger voids which are largely open to the permeate sideof the ultrafilter. Any species capable of passing through the pores ofthe skin can therefore freely pass through the membrane. For maximumretention of solute, a membrane is selected that has a nominal molecularweight cut-off well below that of the species being retained. Inmacromolecular concentration, the membrane enriches the content of thedesired biological species and provides filtrate cleared of retainedsubstances. Microsolutes are removed convectively with the solvent. Asconcentration of the retained solute increases, the ultrafiltration ratediminishes.

[0150] Diafiltration, or buffer exchange, using ultrafilters is an idealway for removal and exchange of salts, sugars, non-aqueous solventsseparation of free from bound species, removal of material of lowmolecular weight, or rapid change of ionic and pH environments.Microsolutes are removed most efficiently by adding solvent to thesolution being ultrafiltered at a rate equal to the ultrafiltrationrate. This washes microspecies from the solution at constant volume,purifying the retained species. The present invention utilizes adiafiltration step to exchange the buffer of the virus supematant priorto Benzonase® treatment.

[0151] 5. Viral Infection

[0152] The present invention employs, in one example, adenoviralinfection of cells in order to generate therapeutically significantvectors. Typically, the virus will simply be exposed to the appropriatehost cell under physiologic conditions, permitting uptake of the virus.Though adenovirus is exemplified, the present methods may beadvantageously employed with other viral vectors, as discussed below.

[0153] A) Adenovirus

[0154] Adenovirus is particularly suitable for use as a gene transfervector because of its mid-sized DNA genome, ease of manipulation, hightiter, wide target-cel range, and high infectivity. The roughly 36 kBviral genome is bounded by 100-200 base pair (bp) inverted terminalrepeats (ITR), in which are contained cis-acting elements necessary forviral DNA replication and packaging. The early (E) and late (L) regionsof the genome that contain different transcription units are divided bythe onset of viral DNA replication.

[0155] The E1 region (E1A and E1B) encodes proteins responsible for theregulation of transcription of the viral genome and a few cellulargenes. The expression of the E2 region (E2A and E2B) results in thesynthesis of the proteins for viral DNA replication. These proteins areinvolved in DNA replication, late gene expression, and host cell shutoff (Renan, 1990). The products of the late genes (L1, L2, L3, L4 andL5), including the majority of the viral capsid proteins, are expressedonly after significant processing of a single primary transcript issuedby the major late promoter (MLP). The MLP (located at 16.8 map units) isparticularly efficient during the late phase of infection, and all themRNAs issued from this promoter possess a 5′ tripartite leader (TL)sequence which makes them preferred mRNAs for translation.

[0156] In order for adenovirus to be optimized for gene therapy, it isnecessary to maximize the carrying capacity so that large segments ofDNA can be included. It also is very desirable to reduce the toxicityand immunologic reaction associated with certain adenoviral products.Elimination of large potions of the adenoviral genome, and providing thedelete gene products in trans, by helper virus and/or helper cells,allows for the insertion of large portions of heterologous DNA into thevector. This strategy also will result in reduced toxicity andimmunogenicity of the adenovirus gene products.

[0157] The large displacement of DNA is possible because the ciselements required for viral DNA replication all are localized in theinverted terminal repeats (ITR) (100-200 bp) at either end of the linearviral genome. Plasmids containing ITR's can replicate in the presence ofa non-defective adenovirus (Hay et al., 1984). Therefore, inclusion ofthese elements in an adenoviral vector should permit replication.

[0158] In addition, the packaging signal for viral encapsidation islocalized between 194-385 bp (0.5-1.1 map units) at the left end of theviral genome (Hearing et al., 1987). This signal mimics the proteinrecognition site in bacteriophage λ DNA where a specific sequence closeto the left end, but outside the cohesive end sequence, mediates thebinding to proteins that are required for insertion of the DNA into thehead structure. E1 substitution vectors of Ad have demonstrated that a450 bp (0-1.25 map units) fragment at the left end of the viral genomecould direct packaging in 293 cells (Levrero et al., 1991).

[0159] Previously, it has been shown that certain regions of theadenoviral genome can be incorporated into the genome of mammalian cellsand the genes encoded thereby expressed. These cell lines are capable ofsupporting the replication of an adenoviral vector that is deficient inthe adenoviral function encoded by the cell line. There also have beenreports of complementation of replication deficient adenoviral vectorsby “helping” vectors, e.g., wild-type virus or conditionally defectivemutants.

[0160] Replication-deficient adenoviral vectors can be complemented, intrans, by helper virus. This observation alone does not permit isolationof the replication-deficient vectors, however, since the presence ofhelper virus, needed to provide replicative functions, would contaminateany preparation. Thus, an additional element was needed that would addspecificity to the replication and/or packaging of thereplication-deficient vector. That element, as provided for in thepresent invention, derives from the packaging function of adenovirus.

[0161] It has been shown that a packaging signal for adenovirus existsin the left end of the conventional adenovirus map (Tibbetts, 1977).Later studies showed that a mutant with a deletion in the E1A (194-358bp) region of the genome grew poorly even in a cell line thatcomplemented the early (E1A) function (Hearing and Shenk, 1983). When acompensating adenoviral DNA (0-353 bp) was recombined into the right endof the mutant, the virus was packaged normally. Further mutationalanalysis identified a short, repeated, position-dependent element in theleft end of the Ad5 genome. One copy of the repeat was found to besufficient for efficient packaging if present at either end of thegenome, but not when moved towards the interior of the Ad5 DNA molecule(Hearing et al., 1987).

[0162] By using mutated versions of the packaging signal, it is possibleto create helper viruses that are packaged with varying efficiencies.Typically, the mutations are point mutations or deletions. When helperviruses with low efficiency packaging are grown in helper cells, thevirus is packaged, albeit at reduced rates compared to wild-type virus,thereby permitting propagation of the helper. When these helper virusesare grown in cells along with virus that contains wild-type packagingsignals, however, the wild-type packaging signals are recognizedpreferentially over the mutated versions. Given a limiting amount ofpackaging factor, the virus containing the wild-type signals arepackaged selectively when compared to the helpers. If the preference isgreat enough, stocks approaching homogeneity should be achieved. /

[0163] B) Retrovirus

[0164] Although adenoviral infection of cells for the generation oftherapeutically significant vectors is a preferred embodiments of thepresent invention, it is contemplated that the present invention mayemploy retroviral infection of cells for the purposes of generating suchvectors. The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains threegenes—gag, pol and env—that code for capsid proteins, polymerase enzyme,and envelope components, respectively. A sequence found upstream fromthe gag gene, termed Y, functions as a signal for packaging of thegenome into virions. Two long terminal repeat (LTR) sequences arepresent at the 5' and 3' ends of the viral genome. These contain strongpromoter and enhancer sequences and are also required for integration inthe host cell genome (Coffm, 1990).

[0165] In order to construct a retroviral vector, a nucleic acidencoding a promoter is inserted into the viral genome in the place ofcertain viral sequences to produce a virus that isreplication-defective. In order to produce virions, a packaging cellline containing the gag, pol and env genes but without the LTR and Ycomponents is constructed (Mann et al., 1983). When a recombinantplasmid containing a human cDNA, together with the retroviral LTR and Ysequences is introduced into this cell line (by calcium phosphateprecipitation for example), the Y sequence allows the RNA transcript ofthe recombinant plasmid to be packaged into viral particles, which arethen secreted into the culture media (Nicolas and Rubenstein, 1988;Temin, 1986; Mann et al., 1983). The media containing the recombinantretroviruses is then collected, optionally concentrated, and used forgene transfer. Retroviral vectors are able to infect a broad variety ofcell types. However, integration and stable expression require thedivision of host cells (Paskind et al., 1975).

[0166] A novel approach designed to allow specific targeting ofretrovirus vectors was recently developed based on the chemicalmodification of a retrovirus by the chemical addition of galactoseresidues to the viral envelope. This modification could permit thespecific infection of cells such as hepatocytes via asialoglycoproteinreceptors, should this be desired.

[0167] A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, the infection of a variety of human cellsthat bore those surface antigens was demonstrated with an ecotropicvirus in vitro (Roux et al., 1989).

[0168] C) Other Viral Vectors

[0169] Other viral vectors may be employed as expression constructs inthe present invention. Vectors derived from viruses such as vacciniavirus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988),adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986;Hermonat and Muzycska, 1984) and herpesviruses may be employed. Theseviruses offer several features for use in gene transfer into variousmammalian cells.

[0170] 6. Engineering of Viral Vectors

[0171] In certain embodiments, the present invention further involvesthe manipulation of viral vectors. Such methods involve the use of avector construct containing, for example, a heterologous DNA encoding agene of interest and a means for its expression, replicating the vectorin an appropriate helper cell, obtaining viral particles producedtherefrom, and infecting cells with the recombinant virus particles. Thegene could simply encode a protein for which large quantities of theprotein are desired, i.e., large scale in vitro production methods.Alternatively, the gene could be a therapeutic gene, for example totreat cancer cells, to express immunomodulatory genes to fight viralinfections, or to replace a gene's function as a result of a geneticdefect. In the context of the gene therapy vector, the gene will be aheterologous DNA, meant to include DNA derived from a source other thanthe viral genome which provides the backbone of the vector. Finally, thevirus may act as a live viral vaccine and express an antigen of interestfor the production of antibodies thereagainst. The gene may be derivedfrom a prokaryotic or eukaryotic source such as a bacterium, a virus, ayeast, a parasite, a plant, or even an animal. The heterologous DNA alsomay be derived from more than one source, Le., a multigene construct ora fusion protein. The heterologous DNA may also include a regulatorysequence which may be derived from one source and the gene from adifferent source.

[0172] A) Therapeudc Genes

[0173] p53 currently is recognized as a tumor suppressor gene(Montenarh, 1992). High levels of mutant p53 have been found in manycells transformed by chemical carcinogenesis, ultraviolet radiation, andseveral viruses, including SV40. The p53 gene is a frequent target ofmutational inactivation in a wide variety of human tumors and is alreadydocumented to be the most frequently-mutated gene in common humancancers (Mercer, 1992). It is mutated in over 50% of human NSCLC(Hollestein et al., 1991) and in a wide spectrum of other tumors.

[0174] The p53 gene encodes a 393-amino-acid phosphoprotein that canform complexes with host proteins such as large-T antigen and EIB. Theprotein is found in normal tissues and cells, but at concentrationswhich are generally minute by comparison with transformed cells or tumortissue. Interestingly, wild-type p53 appears to be important inregulating cell growth and division. Overexpression of wild-type p53 hasbeen shown in some cases to be anti-proliferative in human tumor celllines. Thus, p53 can act as a negative regulator of cell growth(Weinberg, 1991) and may directly suppress uncontrolled cell growth ordirectly or indirectly activate genes that suppress this growth. Thus,absence or inactivation of wild-type p53 may contribute totransformation. However, some studies indicate that the presence ofmutant p53 may be necessary for full expression of the transformingpotential of the gene.

[0175] Wild-type p53 is recognized as an important growth regulator inmany cell types. Missense mutations are common for the p53 gene and areknown to occur in at least 30 distinct codons, often creating dominantalleles that produce shifts in cell phenotype without a reduction tohomozygosity. Additionally, many of these dominant negative allelesappear to be tolerated in the organism and passed on in the germ line.Various mutant alleles appear to range from minimally dysfinctional tostrongly penetrant, dominant negative alleles (Weinberg, 1991).

[0176] Casey and colleagues have reported that transfection of DNAencoding.wild-type p53 into two human breast cancer cell lines restoresgrowth suppression control in such cells (Casey et al., 1991). A similareffect has also been demonstrated on transfection of wild-type, but notmutant, p53 into human lung cancer cell lines (Takahasi et al., 1992).p53 appears dominant over the mutant gene and will select againstproliferation when transfected into cells with the mutant gene. Normalexpression of the transfected p53 is not detrimental to normal cellswith endogenous wild-type p53. Thus, such constructs might be taken upby normal cells without adverse effects. It is thus proposed that thetreatment of p53-associated cancers with wild-type p53 expressionconstructs will reduce the number of malignant cells or their growthrate. Furthermore, recent studies suggest that some p53 wild-type tumorsare also sensitive to the effects of exogenous p53 expression.

[0177] The major transitions of the eukaryotic cell cycle are triggeredby cyclin-dependent kinases, or CDK's. One CDK, cyclin-dependent kinase4 (CDK4), regulates progression through the G₁ phase. The activity ofthis enzyme may be to phosphorylate Rb at late G₁. The activity of CDK4is controlled by an activating subunit, D-type cyclin, and by aninhibitory subunit, e.g. p16^(INK4),which has been biochemicallycharacterized as a protein that specifically binds to and inhibits CDK4,and thus may regulate Rb phosphorylation (Serrano et al., 1993; Serranoet al., 1995). Since the p16^(INK4) protein is a CDK4 inhibitor(Serrano, 1993), deletion of this gene may increase the activity ofCDK4, resulting in hyperphosphorylation of the Rb protein. pl6 also isknown to regulate the function of CDK6.

[0178] p16^(INK4) belongs to a newly described class of CDK-inhibitoryproteins that also includes p16^(B), p21^(WAF1, CIP1, SD11), andp27^(KIP1). The p16^(INK4) gene maps to 9p21, a chromosome regionfrequently deleted in many tumor types. Homozygous deletions andmutations of the p16^(INK4) gene are frequent in human tumor cell lines.This evidence suggests that the p16^(INK4) gene is a tumor suppressorgene. This interpretation has been challenged, however, by theobservation that the frequency of the p16^(INK4) gene alterations ismuch lower in primary uncultured tumors than in cultured cell lines(Caldas et al., 1994; Cheng et al., 1994; Hussussian et al., 1994; Kambet al., 1994a; Kamb et al., 1994b; Mori et al., 1994; Okamoto et al.,1994; Nobori et al., 1995; Orlow et al., 1994; Arap et al., 1995).Restoration of wild-type p16^(INK4) function by transfection with aplasmid expression vector reduced colony formation by some human cancercell lines (Okamoto, 1994; Arap, 1995).

[0179] C-CAM is expressed in virtually all epithelial cells (Odin andObrink, 1987). C-CAM, with an apparent molecular weight of 105 kD, wasoriginally isolated from the plasma membrane of the rat hepatocyte byits reaction with specific antibodies that neutralize cell aggregation(Obrink, 1991). Recent studies indicate that, structurally, C-CAMbelongs to the immunoglobulin (Ig) superfamily and its sequence ishighly homologous to carcinoembryonic antigen (CEA) (Lin and Guidotti,1989). Using a baculovirus expression system, Cheung et al. (1993a;1993b and 1993c) demonstrated that the first Ig domain of C-CAM iscritical for cell adhesion activity.

[0180] Cell adhesion molecules, or CAMs are known to be involved in acomplex network of molecular interactions that regulate organdevelopment and cell differentiation (Edelman, 1985). Recent dataindicate that aberrant expression of CAMs may be involved in thetumorigenesis of several neoplasms; for example, decreased expression ofE-cadherin, which is predominantly expressed in epithelial cells, isassociated with the progression of several kinds of neoplasms (Edelmanand Crossin, 1991; Frixen et al., 1991; Bussemakers et al., 1992;Matsura et al., 1992; Umbas et al., 1992). Also, Giancotti and Ruoslahti(1990) demonstrated that increasing expression of α₅β₁ integrin by genetransfer can reduce tumorigenicity of Chinese hamster ovary cells invivo. C-CAM now has been shown to suppress tumor growth in vitro and invivo.

[0181] Other tumor suppressors that may be employed according to thepresent invention include RB, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II,zacl, p73, BRCA1, VHL, FCC, MMACI, MCC, p16, p21, p57, C-CAM, p27 andBRCA2. Inducers of apoptosis, such as Bax, Bak, Bcl-X₅, Bik, Bid,Harakiri, Ad E1B, Bad and ICE-CED3 proteases, similarly could find useaccording to the present invention.

[0182] Various enzyme genes are of interest according to the presentinvention. Such enzymes include cytosine dearninase,hypoxanthine-guanine phosphoribosyltransferase, galactose-l-phosphateuridyltransferase, phenylalanine hydroxylase, glucocerbrosidase,sphingomyeliiiase, α-L-iduronidase, glucose-6-phosphate dehydrogenase,HSV thymidine kinase and human thymidine kinase.

[0183] Hormones are another group of gene that may be used in thevectors described herein. Included are growth hormone, prolactin,placental lactogen, luteinizing hormone, follicle-stimulating hormone,chorionic gonadotropin, thyroid-stimulating hormone, leptin,adrenocorticotropin (ACTH), angiotensin I and II, β-endorphin,β-melanocyte stimulating hormone (P-MSH), cholecystokinin, endothelin I,galanin, gastric inhibitory peptide (GIP), glucagon, insulin,lipotropins, neurophysins, somatostatin, calcitonin, calcitonin generelated peptide (CGRP), β-calcitonin gene related peptide, hypercalcemiaof malignancy factor (1-40), parathyroid hormone-related protein(107-139) (PTH-rP), parathyroid hormone-related protein (107-111)(PTH-rP), glucagon-like peptide (GLP-1), pancreastatin, pancreaticpeptide, peptide YY, PHM, secretin, vasoactive intestinal peptide (VIP),oxytocin, vasopressin (AVP), vasotocin, enkephalinamide, metorphinamide,alpha melanocyte stimulating hormone (alpha-MSH), atrial natriureticfactor (5-28) (ANF), amylin, amyloid P component (SAP-1), corticotropinreleasing hormone (CRH), growth hormone releasing factor (GHRH),luteinizing hormone-releasing hormone (LHRH), neuropeptide Y, substanceK (neurokinin A), substance P and thyrotropin releasing hormone (TRH).

[0184] Other classes of genes that are contemplated to be inserted intothe vectors of the present invention include interleukins and cytokines.Interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11 IL-12, GM-CSF and G-CSF.

[0185] Examples of diseases for which the present viral vector would beuseful include, but are not limited to, adenosine deaminase deficiency,human blood clotting factor IX deficiency in hemophilia B, and cysticfibrosis, which would involve the replacement of the cystic fibrosistransmembrane receptor gene. The vectors embodied in the presentinvention could also be used for treatment of hyperproliferativedisorders such as rheumatoid arthritis or restenosis by transfer ofgenes encoding angiogenesis inhibitors or cell cycle inhibitors.Transfer of prodrug activators such as the HSV-TK gene can be also beused in the treatment of hyperploiferative disorders, including cancer.

[0186] B) Antisense constructs

[0187] Oncogenes such as ras, myc, neu, raf erb, src, fims, jun, trk,ret, gsp, hst, bcl and abl also are suitable targets. However, fortherapeutic benefit, these oncogenes would be expressed as an antisensenucleic acid, so as to inhibit the expression of the oncogene.

[0188] The term “antisense nucleic acid” is intended to refer to theoligonucleotides complementary to the base sequences ofoncogene-encoding DNA and RNA. Antisense oligonucleotides, whenintroduced into a target cell, specifically bind to their target nucleicacid and interfere with transcription, RNA processing, transport and/ortranslation. Targeting double-stranded (ds) DNA with oligonucleotideleads to triple-helix formation; targeting RNA will lead to double-helixformation.

[0189] Antisense constructs may be designed to bind to the promoter andother control regions, exons, introns or even exon-intron boundaries ofa gene. Antisense RNA constructs, or DNA encoding such antisense RNAs,may be employed to inhibit gene transcription or translation or bothwithin a host cell, either in vitro or in vivo, such as within a hostanimal, including a human subject. Nucleic acid sequences comprising“cornplementary nucleotides” are those which are capable of base-pairingaccording to the standard Watson-Crick complementarity rules. That is,that the larger purines will base pair with the smaller pyrimidines toform only combinations of guanine paired with cytosine (G:C) and adeninepaired with either thymine (A:T), in the case of DNA, or adenine pairedwith uracil (A:U) in the case of RNA.

[0190] As used herein, the terms “complementary” or “antisensesequences” mean nucleic acid sequences that are substantiallycomplementary over their entire length and have very few basemismatches. For example, nucleic acid sequences of fifteen bases inlength may be termed complementary when they have a complementarynucleotide at thirteen or fourteen positions with only single or doublemismatches. Naturally, nucleic acid sequences which are “completelycomplementary” will be nucleic acid sequences which are entirelycomplementary throughout their entire length and have no basemismatches.

[0191] While all or part of the gene sequence may be employed in thecontext of antisense construction, statistically, any sequence 17 baseslong should occur only once in the human genome and, therefore, sufficeto specify a unique target sequence. Although shorter oligomers areeasier to make and increase in vivo accessibility, numerous otherfactors are involved in determining the specificity of hybridization.Both binding affinity and sequence specificity of an oligonucleotide toits complementary target increases with increasing length. It iscontemplated that oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 or more base pairs will be used. One can readilydetermine whether a given antisense nucleic acid is effective attargeting of the corresponding host cell gene simply by testing theconstructs in vitro to determine whether the endogenous gene's functionis affected or whether the expression of related genes havingcomplementary sequences is affected.

[0192] In certain embodiments, one may wish to employ antisenseconstructs which include other elements, for example, those whichinclude C-5 propyne pyrirnidines. Oligonucleotides which contain C-5propyne analogues of uridine and cytidine have been shown to bind RNAwith high affinity and to be potent antisense inhibitors of geneexpression (Wagner et al., 1993).

[0193] As an alternative to targeted antisense delivery, targetedribozymes may be used. The term “ribozyme” refers to an RNA-based enzymecapable of targeting and cleaving particular base sequences in oncogeneDNA and RNA. Ribozymes can either be targeted directly to cells, in theform of RNA oligo-nucleotides incorporating ribozyme sequences, orintroduced into the cell as an expression construct encoding the desiredribozymal RNA. Ribozymes may be used and applied in much the same way asdescribed for antisense nucleic acids.

[0194] C) Antigensfor Vaccines

[0195] Other therapeutics genes might include genes encoding antigenssuch as viral antigens, bacterial antigens, fungal antigens or parasiticantigens. Viruses include picomavirus, coronavirus, togavirus,flavirviru, rhabdovirus, paramyxovirus, orthomyxovirus, bunyavirus,arenvirus, reovirus, retrovirus, papovavirus, parvovirus, herpesvirus,poxvirus, hepadnavirus, and spongiform virus. Preferred viral targetsinclude influenza, herpes simplex virus 1 and 2, measles, small pox,polio or HIV. Pathogens include trypanosomes, tapeworms, roundworms,helminths, . Also, tumor markers, such as fetal antigen or prostatespecific antigen, may be targeted in this manner. Preferred examplesinclude HIV env proteins and hepatitis B surface antigen. Administrationof a vector according to the present invention for vaccination purposeswould require that the vector-associated antigens be sufficientlynon-immunogenic to enable long term expression of the transgene, forwhich a strong immune response would be desired. Preferably, vaccinationof an individual would only be required infrequently, such as yearly orbiennially, and provide long term immunologic protection against theinfectious agent.

[0196] D) Control Regions

[0197] In order for the viral vector to effect expression of atranscript encoding a therapeutic gene, the polynucleotide encoding thetherapeutic gene will be under the transcriptional control of a promoterand a polyadenylation signal. A “promoter” refers to a DNA sequencerecognized by the synthetic machinery of the host cell, or introducedsynthetic machinery, that is required to initiate the specifictranscription of a gene. A polyadenylation signal refers to a DNAsequence recognized by the synthetic machinery of the host cell, orintroduced synthetic machinery, that is required to direct the additionof a series of nucleotides on the end of the MRNA transcript for properprocessing and trafficking of the transcript out of the nucleus into thecytoplasm for translation. The phrase “under transcriptional control”means that the promoter is in the correct location in relation to thepolynucleotide to control RNA polymerase initiation and expression ofthe polynucleotide.

[0198] The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work, have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7-20 bp of DNA, and containing one or more recognitionsites for transcriptional activator or repressor proteins.

[0199] At least one module in each promoter functions to position thestart site for RNA synthesis. The best known example of this is the TATAbox, but in some promoters lacking a TATA box, such as the promoter forthe mammalian terminal deoxynucleotidyl transferase gene and thepromoter for the SV40 late genes, a discrete element overlying the startsite itself helps to fix the place of initiation.

[0200] Additional promoter elements regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the tk promoter, thespacing between promoter elements can be increased to 50 bp apart beforeactivity begins to decline. Depending on the promoter, it appears thatindividual elements can function either cooperatively or independentlyto activate transcription.

[0201] The particular promoter that is employed to control theexpression of a therapeutic gene is not believed to be critical, so longas it is capable of expressing the polynucleotide in the targeted cell.Thus, where a human cell is targeted, it is preferable to position thepolynucleotide coding region adjacent to and under the control of apromoter that is capable of being expressed in a human cell. Generallyspeaking, such a promoter might include either a human or viralpromoter. A list of promoters is provided in the Table 2. TABLE 2PROMOTER Immunoglobulin Heavy Chain Immunoglobulin Light Chain T-CellReceptor HLA DQ α and DQ β β-Interferon Interleukin-2 Interleukin-2Receptor MHC Class II 5 MHC Class II HLA-DRα β-Actin Muscle CreatineKinase Prealbumin (Transthyretin) Elastase I Metallothionein CollagenaseAlbumin Gene α-Fetoprotein τ-Globin β-Globin c-fos c-HA-ras InsulinNeural Cell Adhesion Molecule (NCAM) α1-Antitrypsin H2B (TH2B) HistoneMouse or Type I Collagen Glucose-Regulated Proteins (GRP94 and GRP78)Rat Growth Hormone Human Serum Amyloid A (SAA) Troponin I (TNI)Platelet-Derived Growth Factor Duchenne Muscular Dystrophy SV40 PolyomaRetroviruses Papilloma Virus Hepatitis B Virus Human ImmunodeficiencyVirus Cytomegalovirus Gibbon Ape Leukemia Virus

[0202] The promoter further may be characterized as an induciblepromoter. An inducible promoter is a promoter which is inactive orexhibits low activity except in the presence of an inducer substance.Some examples of promoters that may be included as a part of the presentinvention include, but are not limited to, MT II, MMTV, Colleganse,Stromelysin, SV40, Murine MX gene, α-2-Macroglobulin, MHC class I geneh-2kb, HSP70, Proliferin, Tumor Necrosis Factor, or Thyroid StimulatingHormone a gene. The associated inducers are shown in Table 3. It isunderstood that any inducible promoter may be used in the practice ofthe present invention and that all such promoters would fall within thespirit and scope of the claimed invention. TABLE 3 Element Inducer MT IIPhorbol Ester (TPA) Heavy metals MMTV (mouse mammary tumorGlucocorticoids virus) β-Interferon poly(rI)X poly(rc) Adenovirus 5 E2Ela c-jun Phorbol Ester (TPA), H₂O₂ Collagenase Phorbol Ester (TPA)Stromelysin Phorbol Ester (TPA), IL-1 SV40 Phorbol Ester (TPA) Murine MXGene Interferon, Newcastle Disease Virus GRP78 Gene A23187α-2-Macroglobulin IL-6 Vimentin Serum MHC Class I Gene H-2kB InterferonHSP70 Ela, SV40 Large T Antigen Proliferin Phorbol Ester-TPA TumorNecrosis Factor FMA Thyroid Stimulating Hormone α Thyroid Hormone Gene

[0203] In various embodiments, the human cytomegalovirus (CMV) immediateearly gene promoter, the SV40 early promoter and the Rous sarcoma viruslong terminal repeat can be used to obtain high-level expression of thepolynucleotide of interest. The use of other viral or mammalian cellularor bacterial phage promoters which are well-known in the art to achieveexpression of polynucleotides is contemplated as well, provided that thelevels of expression are sufficient to produce a growth inhibitoryeffect.

[0204] By employing a promoter with well-known properties, the level andpattern of expression of a polynucleotide following transfection can beoptimized. For example, selection of a promoter which is active inspecific cells, such as tyrosinase (melanoma), alpha-fetoprotein andalbumin (liver tumors), CC10 (lung tumor) and prostate-specific antigen(prostate tumor) will permit tissue-specific expression of thetherapeutic gene.

[0205] Enhancers were originally detected as genetic elements thatincreased transcription from a promoter located at a distant position onthe same molecule of DNA. This ability to act over a large distance hadlittle precedent in classic studies of prokaryotic transcriptionalregulation. Subsequent work showed that regions of DNA with enhanceractivity are organized much like promoters. That is, they are composedof many individual elements, each of which binds to one or moretranscriptional proteins.

[0206] The basic distinction between enhancers and promoters isoperational. An enhancer region as a whole must be able to stimulatetranscription at a distance; this need not be true of a promoter regionor its component elements. On the other hand, a promoter must have oneor more elements that direct initiation of RNA synthesis at a particularsite and in a particular orientation, whereas enhancers lack thesespecificities. Promoters and enhancers are often overlapping andcontiguous, often seeming to have a very similar modular organization.

[0207] Additionally any promoter/enhancer combination (as per theEukaryotic Promoter Data Base (EPDB)) could also be used to driveexpression of a particular construct. Use of a T3, T7 or SP6 cytoplasmicexpression system is another possible embodiment. Eukaryotic cells cansupport cytoplasmic transcription from certain bacteriophage promotersif the appropriate bacteriophage polymerase is provided, either as partof the delivery complex or as an additional genetic expression vector.

[0208] Where a cDNA insert is employed, one will typically desire toinclude a polyadenylation signal to effect proper polyadenylation of thegene transcript. The nature of the polyadenylation signal is notbelieved to be crucial to the successful practice of the invention, andany such sequence may be employed. Such polyadenylation signals as thatfrom SV40, bovine growth hormone, and the herpes simplex virus thymidinekinase gene have been found to function well in a number of targetcells.

[0209] 7. Methods of Gene Transfer

[0210] In order to create the helper cell lines of the presentinvention, and to create recombinant adenovirus vectors for usetherewith, various genetic (i.e. DNA) constructs must be delivered to acell. One way to achieve this is via viral transductions usinginfectious viral particles, for example, by transformation with anadenovirus vector of the present invention. Alternatively, retroviral orbovine papilloma virus may be employed, both of which permit permanenttransformation of a host cell with a gene(s) of interest. In othersituations, the nucleic acid to be transferred is not infectious, ie.,contained in an infectious virus particle. This genetic- material mustrely on non-viral methods for transfer.

[0211] Several non-viral methods for the transfer of expressionconstructs into cultured mammalian cells also are contemplated by thepresent invention. These include calcium phosphate precipitation (Grahamand Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990)DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al., 1986;Potter et al., 1984), direct microinjection (Harland and Weintraub,1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al.,1979), cell sonication (Fechheimer et al., 1987), gene bombardment usinghigh velocity microprojectiles (Yang et al., 1990), andreceptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988).

[0212] Once the construct has been delivered into the cell the nucleicacid encoding the therapeutic gene may be positioned and expressed atdifferent sites. In certain embodiments, the nucleic acid encoding thetherapeutic gene may be stably integrated into the genome of the cell.This integration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet furtherembodiments, the nucleic acid may be stably maintained in the cell as aseparate, episomal segment of DNA. Such nucleic acid segments or“episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle. How the expression construct is delivered to a cell and where inthe cell the nucleic acid remains is dependent on the type of expressionconstruct employed.

[0213] In one embodiment of the invention, the expression construct maysimply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be performed by any of the methods mentioned above whichphysically or chemically permeabilize the cell membrane. This isparticularity applicable for transfer in vitro, however, it may beapplied for in vivo use as well. Dubensky et al. (1984) successfullyinjected polyomavirus DNA in the form of CaPO₄ precipitates into liverand spleen of adult and newborn mice demonstrating active viralreplication and acute infection. Benvenisty and Neshif (1986) alsodemonstrated that direct intraperitoneal injection of CaPO₄ precipitatedplasmids results in expression of the transfected genes. It isenvisioned that DNA encoding a CAM may also be transferred in a similarmanner in vivo and express CAM.

[0214] Another embodiment of the invention for transferring a naked DNAexpression construct into cells may involve particle bombardment. Thismethod depends on the ability to accelerate DNA coated microprojectilesto a high velocity allowing them to pierce cell membranes and entercells without killing them (Klein et al., 1987). Several devices foraccelerating small particles have been developed. One such device relieson a high voltage discharge to generate an electrical current, which inturn provides the motive force (Yang et al., 1990). The microprojectilesused have consisted of biologically inert substances such as tungsten orgold beads.

[0215] In a further embodiment of the invention, the expressionconstruct may be entrapped in a liposome. Liposomes are vesicularstructures characterized by a phospholipid bilayer membrane and an inneraqueous medium. Multilamellar liposomes 5 have multiple lipid layersseparated by aqueous medium. They form spontaneously when phospholipidsare suspended in an excess of aqueous solution. The lipid componentsundergo self-rearrangement before the formation of closed structures andentrap water and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991).

[0216] Liposome-mediated nucleic acid delivery and expression of foreignDNA in vitro has been very successful. Using the P-lactamase gene, Wonget aL (1980) demonstrated the feasibility of liposome-mediated deliveryand expression of foreign DNA in cultured chick embryo, HeLa, andhepatoma cells. Nicolau et al. (1987) accomplished successfulliposome-mediated gene transfer in rats after intravenous injection.Also included are various commercial approaches involving “lipofection”technology.

[0217] In certain embodiments of the invention, the liposome may becomplexed with a hemagglutinating virus (HVJ). This has been shown tofacilitate fusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments,the liposome may be complexed or employed in conjunction with nuclearnonhistone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, the liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In that such expression constructshave been successfully employed in transfer and expression of nucleicacid in vitro and in vivo, then they are applicable for the presentinvention.

[0218] Other expression constructs which can be employed to deliver anucleic acid encoding a therapeutic gene into cells arereceptor-mediated delivery vehicles. These take advantage of theselective uptake of macromolecules by receptor-mediated endocytosis inalmost all eukaryotic cells. Because of the cell type-specificdistribution of various receptors, the delivery can be highly specific(Wu and Wu, 1993).

[0219] Receptor-mediated gene targeting vehicles generally consist oftwo components: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. The,most extensively characterized ligands are asialoorosomucoid (ASOR) (Wuand Wu, 1987) and transferring (Wagner et al., 1990). Recently, asynthetic neoglycoprotein, which recognizes the same receptor as ASOR,has been used as a gene delivery vehicle (Ferkol et al., 1993; Peraleset al., 1994) and epidermal growth factor (EGF) has also been used todeliver genes to squamous carcinoma cells (Myers, EPO 0273085).

[0220] In other embodiments, the delivery vehicle may comprise a ligandand a liposome. For example, Nicolau et aL (1987) employedlactosyl-ceramide, a galactose-terminal asialganglioside, incorporatedinto liposomes and observed an increase in the uptake of the insulingene by hepatocytes. Thus, it is feasible that a nucleic acid encoding atherapeutic gene also may be specifically delivered into a cell typesuch as prostate, epithelial or tumor cells, by any number ofreceptor-ligand systems with or without liposomes. For example, thehuman prostate-specific antigen (Watt et al., 1986) may be used as thereceptor for mediated delivery of a nucleic acid in prostate tissue.

[0221] 8. Removing Nucleic Acid Contaminants

[0222] The present invention employs nucleases to remove contaminatingnucleic acids. Exemplary nucleases include Benzonase®, Pulmozyme®; orany other DNase or RNase commonly used within the art.

[0223] Enzymes such as Benzonaze® degrade nucleic acid and have noproteolytic activity. The ability of Benzonase® to rapidly hydrolyzenucleic acids makes the enzyme ideal for reducing cell lysate viscosity.It is well known that nucleic acids may adhere to cell derived particlessuch as viruses. The adhesion may interfere with separation due toagglomeration, change in size of the particle or change in particlecharge, resulting in little if any product being recovered with a givenpurification scheme. Benzonase is well suited for reducing the nucleicacid load during purification, thus eliminating the interference andimproving yield.

[0224] As with all endonucleases, Benzonases® hydrolyzes internalphosphodiester bonds between specific nucleotides. Upon completedigestion, all free nucleic acids present in solution are reduced tooligonucleotides 2 to 4 bases in length.

[0225] 9. Purification Techniques

[0226] The present invention employs a number of different purificationto purify adenoviral vectors of the present invention. Such techniquesinclude those based on sedimentation and chromatography and aredescribed in more detail herein below.

[0227] A) Density Gradient Centrifugation

[0228] There are two methods of density gradient centrifugation, therate zonal technique and the isopycnic (equal density) technique, andboth can be used when the quantitative separation of all the componentsof a mixture of particles is required. They are also used for thedetermination of buoyant densities and for the estimation ofsedimentation coefficients.

[0229] Particle separation by the rate zonal technique is based upondifferences in size or sedimentation rates. The technique involvescarefully layering a sample solution on top of a performed liquiddensity gradient, the highest density of which exceeds that of thedensest particles to be separated. The sample is then centrifuged untilthe desired degree of separation is effected, i.e., for sufficient timefor the particles to travel through the gradient to form discrete zonesor bands which are spaced according to the relative velocities of theparticles. Since the technique is time dependent, centrifugation must beterminated before any of the separated zones pellet at the bottom of thetube. The method has been used for the separation of enzymes, hormones,RNA-DNA hybrids, ribosomal subunits, subcellular organelles, for theanalysis of size distribution of samples of polysomes and forlipoprotein fractionations.

[0230] The sample is layered on top of a continuous density gradientwhich spans the whole range of the particle densities which are to beseparated. The maximum density of the gradient, therefore, must alwaysexceed the density of the most dense particle. During centrifugation,sedimentation of the particles occurs until the buoyant density of theparticle and the density of the gradient are equal (i.e., whereP_(p)=P_(m) in equation 2.12). At this point no further sedimentationoccurs, irrespective of how long centrifugation continues, because theparticles are floating on a cushion of material that has a densitygreater than their own.

[0231] Isopycnic centrifugation, in contrast to the rate zonaltechnique, is an equilibrium method, the particles banding to form zoneseach at their own characteristic buoyant density. In cases where,perhaps, not all the components in a mixture of particles are required,a gradient range can be selected in which unwanted components of themixture will sediment to the bottom of the centrifuge tube whilst theparticles of interest sediment to their respective isopycnic positions.Such a technique involves a combination of both the rate zonal andisopycnic approaches.

[0232] Isopycnic centrifugation depends solely upon the buoyant densityof the particle and not its shape or size and is independent of time.Hence soluble proteins, which have a very similar density (e.g., p=1.3 gcm⁻³ in sucrose solution), cannot usually be separated by this method,whereas subcellular organelles (e.g., Golgi apparatus, p=1.11 g cm⁻³ ,mitochondria, p=1.19 g cm⁻³ and peroxisomes, p=1.23 g cm⁻³ in sucrosesolution) can be effectively separated.

[0233] As an alternative to layering the particle mixture to beseparated onto a preformed gradient, the sample is initially mixed withthe gradient medium to give a solution of uniform density, the gradient‘self-forming’, by sedimentation equilibrium, during centrifugation. Inthis method (referred to as the equilibrium isodensity method), use isgenerally made of the salts of heavy metals (e.g., caesium or rubidium),sucrose, colloidal silica or Metrizamide.

[0234] The sample (e.g., DNA) is mixed homogeneously with, for example,a concentrated solution of caesium chloride. Centrifugation of theconcentrated caesium chloride solution results in the sedimentation ofthe CsCl molecules to form a concentration gradient and hence a densitygradient. The sample molecules (DNA), which were initially uniformlydistributed throughout the tube now either rise or sediment until theyreach a region where the solution density is equal to their own buoyantdensity, i.e. their isopycnic position, where they will band to formzones. This technique suffers from the disadvantage that often very longcentrifugation times (e.g., 36 to 48 hours) are required to establishequilibrium. However, it is commonly used in analytical centrifugationto determine the buoyant density of a particle, the base composition ofdouble stranded DNA and to separate linear from circular forms of DNA.

[0235] Many of the separations can be improved by increasing the densitydifferences between the different forms of DNA by the incorporation ofheavy isotopes (e.g., ¹⁵N) during biosynthesis, a technique used byLeselson and Stahl to elucidate the mechanism of DNA replication inEsherichia coli, or by the binding of heavy metal ions or dyes such asethidium bromide. Isopycnic gradients have also been used to separateand purify viruses and analyze human plasma lipoproteins.

[0236] B) Chromatography

[0237] In certain embodiments of the invention, it will be desirable toproduce purified adenovirus. Purification techniques are well known tothose of skill in the arL These techniques tend to involve thefractionation of the cellular milieu to separate the adenovirusparticles from other components of the mixture. Having separatedadenoviral particles from the other components, the adenovirus may bepurified using chromatographic and electrophoretic techniques to achievecomplete purification. Analytical methods particularly suited to thepreparation of a pure adenovrial particle of the present invention areion-exchange chromatography, size exclusion chromatography;polyacrylamide gel electrophoresis. A particularly efficientpurification method to be employed in conjunction with the presentinvention is HPLC.

[0238] Certain aspects of the present invention concern thepurification, and in particular embodiments, the substantialpurification, of an adenoviral particle. The term “purified” as usedherein, is intended to refer to a composition, isolatable from othercomponents, wherein the adenoviral particle is purified to any degreerelative to its naturally-obtainable form. A purified adenoviralparticle therefore also refers to an adenoviral component, free from theenvironment in which it may naturally occur.

[0239] Generally, “purified” will refer to an adenoviral particle thathas been subjected to fractionation to remove various other components,and which composition substantially retains its expressed biologicalactivity. Where the term “substantially purified” is used, thisdesignation will refer to a composition in which the particle, proteinor peptide forms the major component of the composition, such asconstituting about 50% or more of the constituents in the composition.

[0240] Various methods for quantifying the degree of purification of aprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity, hereinassessed by a “-fold purification number”. The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification and whetheror not the expressed protein or peptide exhibits a detectable activity.

[0241] There is no general requirement that the adenovirus, always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater -fold purification than thesame technique utilizing a low pressure chromatography system. Methodsexhibiting a lower degree of relative purification may have advantagesin total recovery of protein product, or in maintaining the activity ofan expressed protein.

[0242] Of course, it is understood that the chromatographic techniquesand other purification techniques known to those of skill in the art mayalso be employed to purify proteins expressed by the adenoviral vectorsof the present invention. Ion exchange chromatography and highperformance liquid chromatography are exemplary purification techniquesemployed in the purification of adenoviral particles and are describedin further detail herein below.

[0243] Ion-Exchange Chromatography. The basic principle of ion-exchangechromatography is that the affinity of a substance for the exchangerdepends on both the electrical properties of the material and therelative affinity of other charged substances in the solvent. Hence,bound material can be eluted by changing the pH, thus altering thecharge of the material, or by adding competing materials, of which saltsare but one example. Because different substances have differentelectrical properties, the conditions for release vary with each boundmolecular species. In general, to get good separation, the methods ofchoice are either continuous ionic strength gradient elution or stepwiseelution. (A gradient of pH alone is not often used because it isdifficult to set up a pH gradient without simultaneously increasingionic strength) For an anion exchanger, either pH and ionic strength aregradually increased or ionic strength alone is increased. For a cationexchanger, both pH and ionic strength are increased. The actual choiceof the elution procedure is usually a result of trial and error and ofconsiderations of stability. For example, for unstable materials, it isbest to maintain fairly constant pH.

[0244] An ion exchanger is a solid that has chemically bound chargedgroups to which ions are electrostatically bound; it can exchange theseions for ions in aqueous solution. Ion exchangers can be used in columnchromatography to separate molecules according to charge,; actuallyother features of the molecule are usually important so that thechromatographic behavior is sensitive to the charge density, chargedistribution, and the size of the molecule.

[0245] The principle of ion-exchange chromatography is that chargedmolecules adsorb to ion exchangers reversibly so that molecules can bebound or eluted by changing the ionic environment. Separation on ionexchangers is usually accomplished in two stages: first, the substancesto be separated are bound to the exchanger, using conditions that givestable and tight binding; then the column is eluted with buffers ofdifferent pH, ionic strength, or composition and the components of thebuffer compete with the bound material for the binding sites.

[0246] An ion exchanger is usually a three-dimensional network or matrixthat contains covalently linked charged groups. If a group is negativelycharged, it will exchange positive ions and is a cation exchanger. Atypical group used in cation exchangers is the sulfonic group, SO3⁻. Ifan H⁺ is bound to the group, the exchanger is said to be in the acidform; it can, for example, exchange on H⁺ for one Na⁺ or two H⁺ for oneCa²⁺. The sulfonic acid group is called a strongly acidic cationexchanger. Other commonly used groups are phenolic hydroxyl andcarboxyl, both weakly acidic cation exchangers. If the charged group ispositive—for example, a quaternary amino group——is a strongly basicanion exchanger. The most common weakly basic anion exchangers arearomatic or aliphatic amino groups.

[0247] The matrix can be made of various material. Commonly usedmaterials are dextran, cellulose, agarose and copolymers of styrene andvinylbenzene in which the divinylbenzene both cross-links thepolystyrene strands and contains the charged groups. Table 4 gives thecomposition of many ion exchangers.

[0248] The total capacity of an ion exchanger measures its ability totake up exchangeable groups per milligram of dry weight. This number issupplied by the manufacturer and is important because, if the capacityis exceeded, ions will pass through the column without binding. TABLE 4Matrix Exchanger Functional Group Tradename Dextran Strong CationicSulfopropyl SP-Sephadex Weak Cationic Carboxymethyl CM-Sephadex StrongAnionic Diethyl-(2- QAE-Sephadex hydroxypropyl)- aminoethyl Weak AnionicDiethylaminoethyl DEAE-Sephadex Cellulose Cationic CarboxymethylCM-Cellulose Cationic Phospho P-cel Anionic DiethylaminoethylDEAE-cellulose Anionic Polyethylenimine PEI-Cellulose AnionicBenzoylated- DEAE(BND)- naphthoylated, cellulose deiethylaminoethylAnionic p-Aminobenzyl PAB-cellulose Styrene- Strong Cationic Sulfonicacid AG 50 divinyl- benzene Strong Anionic AG 1 Strong Sulfonic acid +AG 501 Cationic + Tetramethyl- Strong Anionic ammonium Acrylic WeakCationic Carboxylic Bio-Rex 70 Phenolic Strong Cationic Sulfonic acidBio-Rex 40 Expoxyamine Weak Anionic Tertiary amino AG-3

[0249] The available capacity is the capacity under particularexperimental conditions (i.e., pH, ionic strength). For example, theextent to which an ion exchanger is charged depends on the pH (theeffect of pH is smaller with strong ion exchangers). Another factor isionic strength because small ions near the charged groups compete withthe sample molecule for these groups. This competition is quiteeffective if the sample is a macromolecule because the higher diffusioncoefficient of the small ion means a greater number of encounters.Clearly, as buffer concentration increases, competition becomes keener.

[0250] The porosity of the matrix is an important feature because thecharged groups are both inside and outside the matrix and because thematrix also acts as a molecular sieve. Large molecules may be unable topenetrate the pores; so the capacity will decease with increasingmolecular dimensions. The porosity of the polystyrene-based resins isdetermined by the amount of cross-linking by the divinylbenzene(porosity decreases with increasing amounts of divinylbenzene). With theDowex and AG series, the percentage of divinylbenzene is indicated by anumber after an X-hence, Dowex 50-X8 is 8% divinylbenzene

[0251] Ion exchangers come in a variety of particle sizes, called meshsize. Finer mesh means- an increased surface-to-volume ration andtherefore increased capacity and decreased time for exchange to occurfor a given volume of the exchanger. On the other hand, fine mesh meansa slow flow rate, which can increase diffusional spreading. The use ofvery fine particles, approximately 10 μm in diameter and high pressureto maintain an adequate flow is called high-performance or high-pressureliquid chromatography or simply HPLC.

[0252] Such a collection of exchangers having such differentproperties—charge, capacity, porosity, mesh - makes the selection of theappropriate one for accomplishing a particular separation difficult. Howto decide on the type of column material and the conditions for bindingand elution is described in the following Examples.

[0253] There are a number of choice to be made when employing ionexchange chromatography as a technique. The first choice to be made iswhether the exchanger is to be anionic or cationic. If the materials tobe bound to the column have a single charge (i.e., either plus orminus), the choice is clear. However, many substances (e.g., proteins,viruses), carry both negative and positive charges and the net chargedepends on the pH. In such cases, the primary factor is the stability ofthe substance at various pH values. Most proteins have a pH range ofstability (i.e., in which they do not denature) in which they are eitherpositively or negatively charged. Hence, if a protein is stable at pHvalues above the isoelectric point, an anion exchanger should be used;if stable at values below the isoelectric point, a cation exchanger isrequired.

[0254] The choice between strong and weak exchangers is also based onthe effect of pH on charge and stability. For example, if a weaklyionized substance that requires very low or high pH for ionization ischromatographed, a strong ion exchanger is called for because itfunctions over the entire pH range. However, if the substance is labile,weak ion exchangers are preferable because strong exchangers are oftencapable of distorting a molecule so much that the molecule denatures.The pH at which the substance is stable must, of course, be matched tothe narrow range of pH in which a particular weak exchanger is charged.Weak ion exchangers are also excellent for the separation of moleculeswith a high charge from those with a small charge, because the weaklycharged ions usually fail to bind. Weak exchangers also show greaterresolution of substances if charge differences are very small. If amacromolecule has a very strong charge, it may be impossible to elutefrom a strong exchanger and a weak exchanger again may be preferable. Ingeneral, weak exchangers are more useful than strong exchangers.

[0255] The Sephadex and Bio-gel exchangers offer a particular advantagefor macromolecules that are unstable in low ionic strength. Because thecross-links in these materials maintain the insolubility of the matrixeven if the matrix is highly polar, the density of ionizable groups canbe made several times greater than is possible with cellulose ionexchangers. The increased charge density means increased affinity sothat adsorption can be carried out at higher ionic strengths. On theother hand, these exchangers retain some of their molecular sievingproperties so that sometimes molecular weight differences annul thedistribution caused by the charge differences; the molecular sievingeffect may also enhance the separation.

[0256] Small molecules are best separated or matrices with small poresize (high degree of cross-linking) because the available capacity islarge, whereas macromolecules need large pore size. However, except forthe Sephadex type, most ion exchangers do not afford the opportunity formatching the porosity with the molecular weight.

[0257] The cellulose ion exchangers have proved to be the best forpurifying large molecules such as proteins and polynucleotides. This isbecause the matrix is fibrous, and hence all functional groups are onthe surface and available to even the largest molecules. In may caseshowever, beaded forms such as DEAE-Sephacel and DEAE-Biogel P are moreuseful because there is a better flow rate and the molecular sievingeffect aids in separation.

[0258] Selecting a mesh size is always difficult. Small mesh sizeimproves resolution but decreases flow rate, which increases zonespreading and decreases resolution. Hence, the appropriate mesh size isusually determined empirically.

[0259] Because buffers themselves consist of ions, they can alsoexchange, and the pH equilibrium can be affected. To avoid theseproblems, the rule of buffers is adopted: use cationic buffers withanion exchangers and anionic buffers with cation exchangers. Becauseionic strength is a factor in binding, a buffer should be chosen thathas a high buffering capacity so that its ionic strength need not be toohigh. Furthermore, for best resolution, it has been generally found thatthe ionic conditions used to apply the sample to the column (theso-called starting conditions) should be near those used for eluting thecolumn.

[0260] High Performance Liquid Chromatography (HPLC) is characterized bya very rapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainan adequate flow rate. Separation can be accomplished in a matter ofminutes, or at most an hour. Moreover, only a very small volume of thesample is needed because the particles are so small and close-packedthat the void volume is a very small fraction of the bed volume. Also,the concentration of the sample need not be very great because the bandsare so narrow that there is very little dilution of the sample.

[0261] 10. Pharmaceutical Compositions and Formulations

[0262] When purified according to the methods set forth above, the viralparticles of the present invention will be administered, in vitro, exvivo or in vivo is contemplated. Thus, it will be desirable to preparethe complex as a pharmaceutical composition appropriate for the intendedapplication. Generally this will entail preparing a pharmaceuticalcomposition that is essentially free of pyrogens, as well as any otherimpurities that could be harmful to humans or animals. One also willgenerally desire to employ appropriate salts and buffers to render thecomplex stable and allow for complex uptake by target cells.

[0263] Aqueous compositions of the present invention comprise aneffective amount of the expression construct and nucleic acid, dissolvedor dispersed in a pharmaceutically acceptable carrier or aqueous medium.Such compositions can also be referred to as inocula The phrases“pharmaceutically or pharmacologically acceptable” refer to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, or a human, asappropriate. As used herein, “pharmaceutically acceptable carrier”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, its use inthe therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

[0264] Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersionsalso can be prepared in glycerol, liquid polyethylene glycols, andmixtures thereof and in oils. Under ordinary conditions of storage anduse, these preparations contain a preservative to prevent the growth ofmicroorganisms.

[0265] The viral particles of the present invention may include classicpharmaceutical preparations for use in therapeutic regimens, includingtheir administration to humans. Administration of therapeuticcompositions according to the present invention will be via any commonroute so long as the target tissue is available via that route. Thisincludes oral, nasal, buccal, rectal, vaginal or topical. Alternatively,administration will be by orthotopic, intradermal subcutaneous,intramuscular, intraperitoneal, or intravenous injection. Suchcompositions would normally be administered as pharmaceuticallyacceptable compositions that include physiologically acceptablecarriers, buffers or other excipients. For application against tumors,direct intratumoral injection, inject of a resected tumor bed, regional(i.e., lymphatic) or general administration is contemplated. It also maybe desired to perform continuous perfusion over hours or days via acatheter to a disease site, e.g., a tumor or tumor site.

[0266] The therapeutic compositions of the present invention areadvantageously administered in the form of injectable compositionseither as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid prior to injection may also beprepared. These preparations also may be emulsified. A typicalcomposition for such purpose comprises a pharmaceutically acceptablecarrier. For instance, the composition may contain about 100 mg of humanserum albumin per milliliter of phosphate buffered saline. Otherpharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike may be used. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oil and injectable organic esters such asethyloleate. Aqueous carriers include water, alcoholic/aqueoussolutions, saline solutions, parenteral vehicles such as sodiumchloride, Ringer's dextrose, etc. Intravenous vehicles include fluid andnutrient replenishers. Preservatives include antimicrobial agents,anti-oxidants, chelating agents and inert gases. The pH and exactconcentration of the various components the pharmaceutical compositionare adjusted according to well known parameters.

[0267] Additional formulations which are suitable for oraladministration. Oral formulations include such typical excipients as,for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonateand the like. The compositions take the form of solutions, suspensions,tablets, pills, capsules, sustained release formulations or powders.When the route is topical, the form may be a cream, ointment, salve orspray.

[0268] An effective amount of the therapeutic agent is determined basedon the intended goal, for example (i) inhibition of tumor cellproliferation, (ii) elimination or killing of tumor cells, (iii)vaccination, or (iv) gene transfer for long term expression of atherapeutic gene. The term “unit dose” refers to physically discreteunits suitable for use in a subject, each unit containing apredetermined-quantity of the therapeutic composition calculated toproduce the desired responses, discussed above, in association with itsadministration, ie., the appropriate route and treatment regimen. Thequantity to be administered, both according to number of treatments andunit dose, depends on the subject to be treated, the state of thesubject and the result desired. Multiple gene therapeutic regimens areexpected, especially for adenovirus.

[0269] In certain embodiments of the present invention, an adenoviralvector encoding a tumor suppressor gene will be used to treat cancerpatients. Typical amounts of an adenovirus vector used in gene therapyof cancer is 10³-10¹⁵ PFU/dose, (10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹,10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵) wherein the dose may be divided intoseveral injections at different sites within a solid tumor. Thetreatment regimen also may involve several cycles of administration ofthe gene transfer vector over a period of 3-10 weeks. Administration ofthe vector for longer periods of time from months to years may benecessary for continual therapeutic benefit.

[0270] In another embodiment of the present invention, an adenoviralvector encoding a therapeutic gene may be used to vaccinate humans orother mammals. Typically, an amount of virus effective to produce thedesired effect, in this case vaccination, would be administered to ahuman or mammal so that long term expression of the transsgene isachieved and a strong host immune response develops. It is contemplatedthat a series of injections, for example, a primary injection followedby two booster injections, would be sufficient to induce an long termimmune response. A typical dose would be from 10 to 10¹⁵ PFU/injectiondepending on the desired result. Low doses of antigen generally induce astrong cell-mediated response, whereas high doses of antigen generallyinduce an antibody-mediated immune response. Precise amounts of thetherapeutic composition also depend on the judgment of the practitionerand are peculiar to each individual.

[0271] 11. Examples

[0272] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

EXAMPLE 1 Materials and Methods

[0273] A) Cells

[0274] 293 cells (human epithelial embryonic kidney cells) from theMaster Cell Bank were used for the studies.

[0275] B) Media

[0276] Dulbecco's modified Eagle's medium (DMEM, 4.5 g/L glucose)+10%fetal bovine serum (FBS) was used for the cell growth phase. For thevirus production phase, the FBS concentration in DMEM was lowered to 2%.

[0277] C) Virus

[0278] AdCMVp53 is a genetically engineered, replication-incompetenthuman type 5 adenovirus expressing the human wild type p53 protein undercontrol of the cytomegalovirus (CMV) immediate early promoter.

[0279] D) Celligen bioreactor

[0280] A Celligen bioreactor (New Brunswick Scientific, Co. Inc.) with 5L total volume (3.5 L working volume) was used to produce virussupernatant using microcarrier culture. 13 g/L glass coated microcarrier(SoloHill) was used for culturing cells in the bioreactor.

[0281] E) Production of virus supernatant in the Celligen bioreactor

[0282] 293 cells from master cell bank (MCB) were thawed and expandedinto Cellfactories (Nunc). Cells were generally split at a confluence ofabout 85-90%. Cells were inoculated into the bioreactor at aninoculation concentration of 1×10⁵ cells/ml. Cells were allowed toattach to the microcarriers by intermittent agitation. Continuousagitation at a speed of 30 rpm was started 6-8 hr post cell inoculation.Cells were cultured for 7 days with process parameters set at pH=7.20,dissolved oxygen (DO)=60% of air saturation, temperature=37° C. On day8, cells were infected with AdCMVp53 at an MOI of 5. Fifty hr post virusinfection, agitation speed was increased from 30 rpm to 150 rpm tofacilitate cell lysis and release of the virus into the supematant. Thevirus supernatant was harvested 74 hr post-infection. The virussupernatant was then filtered for further concentration/diafiltration.

[0283] F) Cellcube™ bioreactor system

[0284] A Cellcube™ bioreactor system (Corning-Costar) was also used forthe production of AdCMVp53 virus. It is composed of a disposable cellculture module, an oxygenator, a medium recirculation pump and a mediumpump for perfusion. The cell culture module used has a culture surfacearea of 21,550 cm² (1 mer).

[0285] G) Production of virus in the Cellcube™

[0286] 293 cells from master cell bank (MCB) were thawed and expandedinto Cellfactories (Nunc). Cells were generally split at a confluence ofabout 85-90%. Cells were inoculated into the Cellcube™ according to themanufacturer's recommendation. Inoculation cell densities were in therange of 1-1.5×10⁴/cm². Cells were allowed to grow for 7 days at 37° C.under culture conditions of pH=7.20, DO=60% air saturation. Mediumperfusion rate was regulated according to the glucose concentration inthe Cellcube™. One day before viral infection, medium for perfusion waschanged from DMEM+10% FBS to DMEM+2% FBS. On day 8, cells were infectedwith AdCMVp53 virus at a multiplicity of infection (MOI) of 5. Mediumperfusion was stopped for 1 hr immediately after infection then resumedfor the remaining period of the virus production phase. Culture washarvested 45-48 hr post-infection.

[0287] H) Lysis solution

[0288] Tween-20 (Fisher Chemicals) at a concentration of 1% (v/v) in 20mM Tris+0.25 M NaCl+1 mM MgCl₂, pH=7.50 buffer was used to lyse cells atthe end of the virus production phase in the Cellcube™.

[0289] I) Clarification and filtration

[0290] Virus supematant from the Celligen bioreactor and virus solutionfrom the Cellcube™ were first clarified using a depth filter (Preflow,GelmanSciences), then was filtered through a 0.8/0.22 μm filter(SuporCap 100, GelmanSciences).

[0291] J) Concentration/diafiltration

[0292] Tangential flow filtration (TFF) was used to concentrate andbuffer exchange the virus supernatant from the Celligen bioreactor andthe virus solution from the Cellcube™. A Pellicon II mini cassette(Millipore) of 300 K nominal molecular weight cut off (NMWC) was usedfor the concentration and diafiltration. Virus solution was firstconcentrated 10-fold. This was followed by 4 sample volume of bufferexchange against 20 mM Tris+1.0 M NaCl+1 mM MgCl₂, pH=9.00 buffer usingthe constant volume diafiltration method.

[0293] Similar concentration/diafiltration was carried out for thecolumn purified virus. A Pellicon II mini cassette of 100 K NMWC wasused instead of the 300 K NMWC cassette. Diafiltration was done against20 mM Tris+0.25 M NaCl+1 mM MgCl₂, pH=9.00 buffer or Dulbecco'sphosphate buffered saline (DPBS).

[0294] K) Benzonase treatnent

[0295] The concentrated/diafiltrated virus solution was treated withBenzonase™ (American International Chemicals) at a concentration of 100u/ml, room temperature overnight to reduce the contaminating nucleicacid concentration in the virus solution.

[0296] L) CsCl gradient ultracentrifugation

[0297] Crude virus solution was purified using double CsCl gradientultracentrifugation using a SW40 rotor in a Beckman ultracentrifuge(XL-90). First, 7 ml of crude virus solution was overlaid on top of astep CsCl gradient made of equal volume of 2.5 ml of 1.25 g/ml and 1.40g/ml CsCl solution, respectively. The CsCl gradient was centrifuged at35,000 rpm for 1 hr at room temperature. The virus band at the gradientinterface was recovered. The recovered virus was then further purifiedthrough a isopicnic CsCl gradient. This was done by mixing the virussolution with at least 1.5-fold volume of 1.33 g/ml CsCl solution. TheCsCl solution was centrifuged at 35,000 rpm for at least 18 hr at roomtemperature. The lower band was recovered as the intact virus. The viruswas immediately dialyzed against 20 mM Tris+1 mM MgCl₂, pH=7.50 bufferto remove CsCl. The dialyzed virus was stored at −7° C. for future use.

[0298] M) Ion exchange chromatography (IEC) purification

[0299] The Benzonase treated virus solution was purified using IEC.Strong anionic resin Toyopearl SuperQ 650M (Tosohaas) was used for thepurification. A FPLC system (Pharmacia) with a XK16 column (Pharrnacia)were used for the initial method development. Further scale-up studieswere carried out using a BioPilot system (Pharmacia) with a XK 50 column(Pharmacia). Briefly, the resin was packed into the columns andsanitized with 1 N NaOH, then charged with buffer B which was followedby conditioning with buffer A. Buffers A and B were composed of 20 mMTris+0.25 M NaCl+1 mM MgCl₂, pH=9.00 and 20 mM Tris+2M NaCl+1 mM MgCl₂,pH=9.00, respectively. Viral solution sample was loaded onto theconditioned column, followed by washing the column with buffer A untilthe UV absorption reached base line. The purified virus was eluted fromthe column by using a 10 column volume of linear NaCl gradient.

[0300] N) HPLC analysis

[0301] A HPLC analysis procedure was developed for evaluating theefficiency of virus production and purification.Tris(hydroxymethyl)aminomethane (tris) was obtained from FisherBiotech(Cat# BP154-1; Fair Lawn, N.J., U.S.A.); sodium chloride (NaCl) wasobtained from Sigma (Cat# S-7653, St. Louis, Mo., U.S.A.). Both wereused directly without firther purification. HPLC analyses were performedon an Analytical Gradient System from Beckman, with Gold WorkstationSoftware (126 binary pump and 168 diode array detector) equipped with ananion-exchange column from TosoHaas (7.5 cm×7.5 mm ID, 10 μm particlesize, Cat# 18257). A 1-ml Resource Q (Pharmacia) anion-exchange columnwas used to evaluate the method developed by Huyghe et al. using theirHEPES buffer system. This method was only tried for the Bioreactorsystem.

[0302] The buffers used in the present HPLC system were Buffer A: 10 mMtris buffer, pH 9.0. Buffer B: 1.5 M NaCl in buffer A, pH 9.0. Thebuffers were filtered through a 0.22 μm bottle top filter by Corning(Cat# 25970-33). All of the samples were filtered through a 0.8/0.22 μmAcrodisc PF from Gelman Sciences (Cat# 4187) before injection.

[0303] The sample is injected onto the HPLC column in a 60-100 μlvolume. After injection, the column (TosoHaas) is washed with 20% B for3 min at a flow rate of 0.75 ml/min. A gradient is then started, inwhich B is increased from 20% to 50% over 6 min. Then the gradient ischanged from 50% to 100% B over 3 min, followed by 100% B for 6 min. Thesalt concentration is then changed back stepwise to 20% again over 4min, and maintained at 20% B for another 6 min. The retention time ofthe Adp53 is 9.5±0.3 min with A₂₆₀/A₂₈₀≅1.26±0.03. Cleaning of thecolumn after each chromatographic run is accomplished by injecting 100μl of 0.15 M NaOH and then running the gradient.

EXAMPLE 2

[0304] Effect of medium perfusion rate in Cellcube™ on virus productionand purification

[0305] For a perfusion cell culture system, such as the Cellcube™,medium perfusion rate plays an important role on the yield and qualityof product. Two different medium perfusion strategies were examined. Onestrategy was to keep the glucose concentration in the Cellcube™ ≧2 g/L(high perfusion rate). The other one was to keep the glucoseconcentration ≧1 g/L (low medium perfusion rate).

[0306] No significant changes in the culture parameters, such as pH, DO,was observed between the two different perfusion rates. Approximatelyequivalent amount of crude viruses (before purification) were producedafter harvesting using 1% Tween-20 lysis solution as shown in Table 5.However, dramatic difference was seen on the HPLC profiles of the viralsolutions from the high and low medium perfusion rate production runs.TABLE 5 Effect of medium glucose concentration on virus yield Glucoseconcentration (g/L) ≧2.0 ≧1.0 Crude virus yield (PFU) 4 × 10¹² 4.9 ×10¹²

[0307] As shown in FIG. 1, a very well separated virus peak (retentiontime 9.39 min) was produced from viral solution using low mediumperfusion rate. It was found that virus with adequate purity andbiological activity was attained after a single step ion exchangechromatographic purification of the virus solution produced under lowmedium perfusion rate. On the other hand, no separated virus peak in theretention time of 9.39 min was observed from viral solution producedusing high medium perfusion rate. This suggests that contaminants whichhave the same elution profile as the virus were produced under highmedium perfusion rate. Although the nature of the contaminants is notyet clear, it is expected that the contaminants are related to theincreased extracellular matrix protein production under high mediumperfusion rate (high serum feeding) from the producer cells. This poorseparation characteristic seen on the HPLC created difficulties forprocess IEC purification as shown in the following Examples. As aresult, medium perfusion rate used during the cell growth and the virusproduction phases in the Cellcube™ has a significant effect on thedownstream IEC purification of the virus. Low medium perfusion rate isrecommended. This not only produces easy to purify crude product butalso offers more cost-effective production due to the reduced mediumconsumption.

EXAMPLE 3

[0308] Methods of cell harvest and lysis

[0309] Based on previous experience, the inventors first evaluated thefreeze-thaw method. Cells were harvested from the Cellcube™ 4548 hrpost-infection. First, the Cellcube™ was isolated from the culturesystem and the spent medium was drained. Then, 50 mM EDTA solution waspumped into the Cube to detach the cells from the culture surface. Thecell suspension thus obtained was centrifuged at 1,500 rpm (BeckmanGS-6KR) for 10 min. The resultant cell pellet was resuspended inDulbecco's phosphate buffered saline (DPBS). The cell suspension wassubjected to 5 cycles of freeze/thaw between 37° C. water bath anddry-ice ethanol bath to release virus from the cells. The crude celllysate (CCL) thus generated was analyzed on HPLC.

[0310]FIG. 2 shows the HPLC profile. No virus peak is observed atretention time of 9.32 min. Instead, two peaks at retention times of9.11 and 9.78 min are produced. This profile suggests that the othercontaminants having similar elution time as the virus exist in the CCLand interfere with the purification of the virus. As a result, very lowpurification efficiency was observed when the CCL was purified by IECusing FPLC.

[0311] In addition to the low purification efficiency, there was asignificant product loss during the cell harvest step into the EDTAsolution as indicated in Table 6. Approximately 20% of the product waslost into the EDTA solution which was discarded. In addition, about 24%of the crude virus product is present in the spent medium which was alsodiscarded. Thus, only 56% of the crude virus product is in the CCL.Furthermore, freeze-thaw is a process of great variation and verylimited scaleability. A more efficient cell lysis process with lessproduct loss needed to be developed. TABLE 6 Loss of virus during EDTAharvest of cells from Cellcube ™ Crude EDTA product Total Waste harvestCrude cell crude Spent Medium Solution lysate product (PFU) Volume (ml)2800 2000 82 — Titer 2.6 × 10⁸  3 × 10⁸    2 × 10¹⁰ — (PFU/ml) Total 7.2× 10¹¹ 6 × 10¹¹ 1.64 × 10¹² 3 × 10¹² virus (PFU) Percentage 24% 20% 56%

[0312] TABLE 7 Evaluation of non-ionic detergents for cell lysis Concen-trations Detergents (w/v) Chemistry Comments Thesit   1% Dodecylpoly-Large (ethylene glycol ether)_(n), Precipitate 0.5% n = 9-10 0.1% NP-40  1% Ethylphenolpoly Large (ethylene-glycolether)_(n) precipitate 0.5% n= 9-11 0.1% Tween-20   1% Poly- Small (oxyethylene)_(n)- precipitatesorbitan-monolaurate 0.5% n = 20 0.1% Brij-58   1% Cloudy 0.5% CetylpolySolution 0.1% (ethyleneglycolether)_(n) n = 20 Triton X-100   1%Octylphenolpoly- Large (ethyleneglycolether)_(n) precipitate 0.5% n = 100.1%

[0313] Detergents have been used to lyse cells to release intracellularorganelles. Consequently, the inventors evaluated the detergent lysismethod for the release of adenovirus. Table 7 lists the 5 differentnon-ionic detergents that were evaluated for cell lysis. Cells wereharvested from the Cellcube™ 48 hr post-infection using 50 mM EDTA. Thecell pellet was resuspended in the different detergents at variousconcentrations listed in Table 7.

[0314] Cell lysis was carried out at either room temperature or on icefor 30 min. Clear lysis solution was obtained after centrifugation toremove the precipitate and cellular debris. The lysis solutions weretreated with Benzonase and then analyzed by HPLC. FIG. 3 shows the HPLCprofiles of lysis solutions from the different detergents. Thesit andNP40 performed similarly as Triton X-100. Lysis solution generated from1% Tween-20 gave the best virus resolution with the least virusresolution being observed with Brij-58. More efficient cell lysis wasfound at detergent concentration of 1% (w/v). Lysis temperature did notcontribute significantly to the virus resolution under the detergentconcentrations examined. For the purpose of process simplicity, lysis atroom temperature is recommended. Lysis solution composed of 1% Tween-20in 20 mM Tris +0.25M NaCl+1 mM MgCl₂, pH=7.50 was employed for celllysis and virus harvest in the Cellcube™.

EXAMPLE 4

[0315] Effects of concentrationldiafiltration on virus recovery

[0316] Virus solution from the lysis step was clarified and filteredbefore concentration/ diafiltration. TFF membranes of different NMWCs,including 100K, 300K, 500K, and 1000K, were evaluated for efficientconcentrationldiafiltration. The highest medium flux with minimal virusloss to the filtrate was obtained with a membrane of 300K NMWC. BiggerNMWC membranes offered higher medium flux, but resulted in greater virusloss to the filtrate, while smaller NMWC membranes achieved aninsufficient medium flux. Virus solution was first concentrated 10-fold,which was followed by 4 sample volumes of diafiltration against 20 mMTris+0.25 M NaCl+1 mM MgCl₂, pH=9.00 buffer using the constant volumemethod. During the concentration/diafiltration process, pressure dropacross the membrane was kept ≦5 psi. Consistent, high level virusrecovery was demonstrated during the concentration/diafiltration step asindicated in Table 8. TABLE 8 Concentration/diafiltration of crude virussolution Titer (PFU/ml) Volume (ml) Total virus (PFU) Recovery Run #1Run #2 Run #1 Run #2 Run #1 Run #2 Run #1 Run #2 Before 2.6 × 10⁹ 2 ×10⁹ 1900 2000 4.9 × 10¹² 4 × 10¹² conc./diafl. Post  2.5 × 10¹⁰ 1.7 ×10¹⁰  200 200   5 × 10¹² 3.4 × 10¹²   102% 85% conc./diafl. Conc. 9.5 10Factor Filtrate   5 × 10⁵ 1 × 10⁶ 3000 3000 1.5 × 10⁹  3 × 10⁹ 

EXAMPLE 5

[0317] Effect of salt addition on Benzonase treatment

[0318] Virus solution after concentration/diafiltration was treated withBenzonase (nuclease) to reduce the concentration of contaminatingnucleic acid in virus solution. Different working concentrations ofBenzonase, which included 50, 100, 200, 300 units/ml, were evaluated forthe reduction of nucleic acid concentrations. For the purpose of processsimplicity, treatment was carried out at room temperature overnight.Significant reduction in contaminating nucleic acid that is hybridizableto human genomic DNA probe was seen after Benzonase treatment.

[0319] Table 9 shows the reduction of nucleic acid concentration beforeand after Benzonase treatment. Virus solution was analyzed on HPLCbefore and after Benzonase treatment. As shown in FIG. 4A and FIG. 4B,dramatic reduction in the contaminating nucleic acid peak was observedafter Benzonase treatment. This is in agreement with the result of thenucleic acid hybridization assay. Because of the effectiveness, aBenzonase concentration of 100 ulml was employed for the treatment ofthe crude virus solution. TABLE 9 Reduction of contaminating nucleicacid concentration in virus solution Before Treatment After TreatmentReduction Contaminating 200 μg/ml 10 ng/ml 2 × 10⁴-fold nucleic acidconcentration

[0320] Considerable change in the HPLC profile was observed pre- andpost-Benzonase treatment. No separated virus peak was detected atretention time of 9.33 min after Benzonase treatment. At the same time,a major peak with high 260 nm adsorption at retention time of 9.54 minwas developed. Titer assay results indicated that Benzonase treatmentdid not negatively affect the virus titer and virus remained intact andinfectious after Benzonase treatment. It was reasoned that cellularnucleic acid released during the cell lysis step interacted with virusand either formed aggregates with the virus or adsorbed onto the virussurface during Benzonase treatment.

[0321] To minimize the possible nucleic acid virus interaction duringBenzonase treatment, different concentrations of NaCl was added into thevirus solution before Benzonase treatment. No dramatic change in theHPLC profile occurred after Benzonase treatment in the presence of 1 MNaCl in the virus solution. FIG. 5 shows the HPLC profile of virussolution after Benzonase treatment in the presence of 1M NaCl. Unlikethat shown in FIG. 4B, virus peak at retention time of 9.35 min stillexists post Benzonase treatment. This result indicates that the presenceof 1M NaCl prevents the interaction of nucleic acid with virus duringBenzonase treatment and facilitates the further purification of virusfrom contaminating nucleic acid.

EXAMPLE 6

[0322] Ion exchange chromatographic purification

[0323] The presence of negative charge on the surface of adenovirus atphysiological pH conditions prompted evaluation of anionic ionexchangers for adenovirus purification. The strong anionic ion exchangerToyopearl Super Q 650M was used for the development of a purificationmethod. The effects of NaCl concentration and pH of the loading buffer(buffer A) on virus purification was evaluated using the FPLC system.

[0324] A) Method development

[0325] For ion exchange chromatography, buffer pH is one of the mostimportant parameters and can have dramatic influence on the purificationefficiency. In reference to the medium pH and conductivity used duringvirus production, the inventors formulated 20 mM Tris+1 mM MgCl₂+0.2MNaCl, pH=7.50 as buffer A. A XK16 column packed with Toyopearl SuperQ650M with a height of 5 cm was conditioned with buffer A.

[0326] A sample of 5 ml of Benzonase treated concentratedldiafiltratedvirus supematant from the Celligen bioreactor was loaded onto thecolumn. After washing the column, elution was carried out with a lineargradient of over 10 column volumes of buffer B formulation to reach mMTris+1 mM MgCl₂ +2M NaCl, pH=7.50.

[0327]FIG. 6 shows the elution profile. Three peaks were observed duringelution without satisfactory separation among them. Control studyperformed with 293 cell conditioned medium (with no virus) showed thatthe first two peaks are virus related. To further improve the separationefficiency, the effect of buffer pH was evaluated. Buffer pH wasincreased to 9.00 while keeping other conditions constant. Much improvedseparation, as shown in FIG. 7, was observed as compared to that ofbuffer pH of 7.50. Fractions #3, #4, and #8 were analyzed on HPLC.

[0328] As shown in FIG. 8, the majority of virus was found in fraction#4, with no virus being detected in fractions #3 and #8. Fraction #8 wasfound to be mainly composed of contaminating nucleic acid However, thepurification was still not optimal. There is overlap between fractions#3 and #4 with contaminants still detected in fraction #4.

[0329] Based on the chromatogram in FIG. 7, it was inferred that furtherimprovement of virus purification could be achieved by increasing thesalt concentration in buffer A. As a result, the contaminants present inthe fraction #3, which is prior to the virus peak, can be shifted to theflow through faction. The NaCl concentration in buffer A was increasedto 0.3 M while keeping other conditions constant. FIG. 9 shows theelution profile under the condition of 0.3 M NaCl in buffer A.

[0330] Dramatic improvement in purification efficiency was achieved. Asexpected the contaminant peak observed in FIG. 7 was eliminated underthe increased salt condition. Samples from crude virus sup, flowthrough, peak #1, and peak #2 were analyzed on HPLC and the results areshown in FIG. 10. No virus was detected in the flow through fraction.The majority of the contaminants present in the crude material werefound in the flow through. HPLC analysis of peak #1 showed a single welldefined virus peak. This HPLC profile is equivalent to that obtainedfrom double CsCl gradient purified virus. Peaks observed at retentiontimes of 3.14 and 3.61 min in CsCl gradient purified virus are glycerolrelated peaks. The purified virus has a A260/A280 ratio of 1.27±0.03.This similar to the value of double CsCl gradient purified virus as wellas the results reported by Huyghe et al. (1996). Peak #2 is composedmainly of contaminating nucleic acid. Based on the purification result,the inventors proposed the following method for IEC purification ofadenovirus sup from the bioreactor.

[0331] Buffer A: 20 mM Tris+1 mM MgCl₂ +0.3M NaCl, pH=9.00

[0332] Buffer B: 20 mM Tris+1 mM MgCl₂ +2M NaCl, pH=9.00

[0333] Elution: 10 column volume linear gradient

[0334] B) Method scale-up

[0335] Following the development of the method, purification wasscaled-up from the XK16 column (1.6 cm I.D.) to a XK50 column (5cmI.D.,10-fold scale-up) using the same purification method. A similarelution profile was achieved on the XK50 column as shown in FIG. 11. Thevirus fraction was analyzed on HPLC, which indicated equivalent viruspurity to that obtained from the XK16 column.

[0336] During the scale-up studies, it was found that it was moreconvenient and consistent to use conductivity to quantify the saltconcentration in buffer A. The optimal conductivity of buffer A is inthe range of 25±2 mS/cm at approximately room temperature (21° C.).Samples produced during the purification process together with doubleCsCl purified virus were analyzed on SDS-PAGE.

[0337] As shown in FIG. 12, all the major adenovirus structure proteinsare detected on the SDS-PAGE. The IEC purified virus shows equivalentstaining as that of the double CsCl purified virus. Significantreduction in bovine serum albumin (BSA) concentration was achievedduring purification. The BSA concentration in the purified virus wasbelow the detection level of the western blot assay as shown in FIG. 13.

[0338] The reduction of contaminating nucleic acid concentration invirus solution during the purification process was determined usingnucleic acid slot blot. ³²P labeled human genomic DNA was used as thehybridization probe (because 293 cells are human embryonic kidneycells). Table 10 shows the nucleic acid concentration at differentstages of the purification process. Nucleic acid concentration in thefinal purified virus solution was reduced to 60 pg/ml, an approximate3.6×10⁶-fold reduction compared to the initial virus supernatant. Virustiter and infectious to total particle ratio were determined for thepurified virus and the results were compared to that from double CsClpurification in Table 9. Both virus recovery and particle/PFU ratio arevery similar between the two purification methods. The titer of thecolumn purified virus solution can be further increased by performing aconcentration step. TABLE 10 Removal of contaminating nucleic acidsduring purification Contaminating nucleic acid Steps during purificationconcentration Virus supernatant from bioreactor 220 μg/mlConcentrated/diafiltrated sup 190 μg/ml Sup post Benzonase treatment(O/N, RT, 10 ng/ml 100 u/ml) Purified virus from column 210 pg/mlPurified virus post 60 pg/ml concentration/diafiltration CsCl purifiedvirus 800 pg/ml

EXAMPLE 7

[0339] Other purification methods

[0340] In addition to the strong anionic ion exchange chromatography,other modes of chromatographic methods, were also evaluated for thepurification of AdCMVpS3 virus (e.g. size exclusion chromatography,hydrophobic interaction chromatography, cation exchange chromatography,or metal ion affinity chromatography). Compared to the Toyopearl SuperQ, all those modes of purification offered much less efficientpurification with low product recovery. Therefore, Toyopearl Super Qresin is recommended for the purification of AdCMVpS3. However, otherquaternary ammonium chemistry based strong anionic exchangers are likelyto be suitable for the purification of AdCMVp53 with some processmodifications.

EXAMPLE 8

[0341] Purification of crude AdCMVp53 virus generated from Cellcube™

[0342] Two different production methods were developed to produceAdCMVp53 virus. One was based on microcarrier culture in a stirred tankbioreactor. The other was based on a Cellcube™ bioreactor. As describedabove, the purification method was developed using crude virussupematant generated from the stirred tank bioreactor. It was realizedthat although the same medium, cells and viruses were used for virusproduction in both the bioreactor and the Cellcube™, the culture surfaceonto which cells attached was different.

[0343] In the bioreactor, cells were grown on a glass coatedmicrocarrier, while in the Cellcube™ celis were grown on proprietarytreated polystyrene culture surface. Constant medium perfusion was usedin the Cellcube™, on the other hand, no medium perfusion was used in thebioreactor. In the Cellcube™, the crude virus product was harvested inthe form of virally infected cells, which is different from the virussupernatant harvested from the bioreactor.

[0344] Crude cell lysate (CCL), produced after 5 cycles freeze-thaw ofthe harvested virally infected cells, was purified by IEC using theabove described method. Unlike the virus supernatant from thebioreactor, no satisfactory purification was achieved for the CCLmaterial generated from the Cellcube™. FIG. 14 shows the chromatogram.The result suggests that crude virus solution generated from theCellcube™ by freeze-thawing harvested cells is not readily purified bythe IEC method.

[0345] Other purification methods, including hydrophobic interaction andmetal chelate chromatography, were examined for the purification ofvirus in CCL. Unfortunately, no improvement in purification was observedby either method. Considering the difficulties of purification of virusin CCL and the disadvantages associated with a freeze-thaw step in theproduction process, the inventors decided to explore other cell lysismethods.

[0346] A) Purification of crude virus solution in lysis buffer

[0347] As described in Examples 1 and 3, HPLC analysis was used toscreen different detergent lysis methods. Based on the HPLC results, 1%Tween-20 in 20 mM Tris+0.25 M NaCl+1 mM MgCl₂, pH=7.50 buffer wasemployed as the lysis buffer. At the end of the virus production phase,instead of harvesting the infected cells, the lysis buffer was pumpedinto the Cellcube™ after draining the spent medium. Cells were lysed andvirus released into the lysis buffer by incubating for 30 min.

[0348] After clarification and filtration, the virus solution wasconcentrated/diafiltrated and treated with Benzonase to reduce thecontaminating nucleic acid concentration. The treated virus solution waspurified by the method developed above using Toyopearl SuperQ resin.Satisfactory separation, similar to that obtained using virussupernatant from the bioreactor, was achieved during elution. FIG. 15shows the elution profile. However, when the virus fraction was analyzedon HPLC, another peak in addition to the virus peak was detected. Theresult is shown in FIG. 16A.

[0349] To further purify the virus, the collected virus fraction wasre-purified using the same method. As shown in FIG. 16B, purity of thevirus fraction improved considerably after the second purification.Metal chelate chromatography was also evaluated as a candidate for thesecond purification. Similar improvement in virus purity as seen withthe second IEC was achieved. However, because of its simplicity, IEC ispreferred as the method of choice for the second purification.

[0350] As described above in Example 2, medium perfusion rate employedduring the cell growth and virus production phases has a considerableimpact on the HPLC separation profile of the Tween-20 crude virusharvest. For crude virus solution produced under high medium perfusionrate, two ion exchange columns are required to achieve the requiredvirus purity.

[0351] Based on the much improved separation observed on HPLC for virussolution produced under low medium perfusion rate, it is likely thatpurification through one ion exchange column may achieve the requiredvirus purity. FIG. 17 shows the elution profile using crude virussolution produced under low medium perfusion rate. A sharp virus peakwas attained during elution. HPLC analysis of the virus fractionindicates virus purity equivalent to that of CsCl gradient purifiedvirus after one ion exchange chromatography step. FIG. 18 shows the HPLCanalysis result.

[0352] The purified virus was further analyzed by SDS-PAGE, western blotfor BSA, and nucleic acid slot blot to determine the contaminatingnucleic acid concentration. The analysis results are given in FIG. 19A,FIG. 19B and FIG. 19C, respectively. All those analyses indicate thatthe column purified virus has equivalent purity compared to the doubleCsCl gradient purified virus. Table 11 shows the virus titer andrecovery before and after the column purification. For comparisonpurposes, the typical virus recovery achieved by double CsCl gradientpurification was also included. Similar virus recoveries were achievedby both methods. TABLE 11 Comparison of IEC and double CsCl gradientultracentrifugation purification of AdCMVp53 from Cellcube ™ Titer A260/Particle/ (PFU/ml) A280 PFU Recovery IEC 1 × 10¹⁰ 1.27 36 63%Ultracentrifugation 2 × 10¹⁰ 1.26 38 60%

[0353] A) Resin capacity study

[0354] The dynamic capacity of the Toyopearl Super Q resin was evaluatedfor the purification of the Tween-20 harvested virus solution producedunder low medium perfusion rate. One hundred ml of resin was packed in aXK50 column. Different amount of crude virus solution was purifiedthrough the column using the methods described herein.

[0355] Virus breakthrough and purification efficiency were analyzed onHPLC. FIG. 20 shows the HPLC analysis results. At a column loadingfactor greater than sample/column volume ratio of 2:1, purity of thevirus fraction was reduced. Contaminants co-eluted with the virus. At aloading factor of greater than 3:1, breakthrough of the virus into theflow through was observed. Therefore, it was proposed that the workingloading capacity of the resin be in the range of sample/column volumeratio of 1:1.

[0356] B) Concentration/diafiltration post purificadon

[0357] A concentrationldiafiltration step after column purificationserves not only to increase the virus titer, if necessary, but also toexchange to the buffer system specified for the virus product. A 300KNMWC TFF membrane was employed for the concentration step. Because ofthe absence of proteinacious and nucleic acid contaminants in thepurified virus, very high buffer flux was achieved without noticeablepressure drop across the membrane.

[0358] Approximately 100% virus recovery was achieved during this stepby changing the buffer into 20 mM Tris+1 mM MgCl₂ +0.15 M NaCl, pH=7.50.The purified virus was also successfully buffer exchanged into DPBSduring the concentration/diafiltration step. The concentration factorcan be determined by the virus titer that is desired in the finalproduct and the titer of virus solution eluted from the column. Thisflexibility will help to maintain the consistency of the final purifiedvirus product.

[0359] C) Evaluation of defective adenovirus in the IEC purifiedAdCMVp53

[0360] Due to the less than 100% packaging efficiency of adenovirus inproducer cells, some defective adenoviruses generally exist in crudevirus solution. Defective viruses do not have DNA packaged inside theviral capsid and therefore can be separated from intact virus on CsClgradient ultracentrifugation based the density difference. It is likelythat it would be difficult to separate the defective from the intactviruses based on ion exchange chromatography assuming both viruses havesimilar surface chemistry. The presence of excessive amount of defectiveviruses will impact the quality of the purified product.

[0361] To evaluate the percentage of defective virus particles present,the purified and concentrated viruses were subjected to isopicnic CsClultracentrifugation. As shown in FIG. 21, a faint band on top of theintact virus band was observed after centrifugation. Both bands wererecovered and dialyzed against 20 mM Tris+1 MM MgCl₂, pH=7.50 buffer toremove CsCl. The dialyzed viruses were analyzed on HPLC and the resultsare shown in FIG. 22. Both viruses show similar retention time. However,the defective virus has a smaller A260/A280 ratio than that of theintact virus. This is indicative of less viral DNA in the defectivevirus.

[0362] The peaks seen at retention times between 3.02 to 3.48 min areproduced by glycerol which is added to the viruses (10% v/v) beforefreezing at −70° C. The percentage of the defective virus was less than1% of the total virus. This low percentage of defective virus isunlikely to impact the total particle to infectious virus (PFU) ratio inthe purified virus product. Both viruses were analyzed by SDS-PAGE(shown in FIG. 19A). Compared to the intact viruses, defective viruseslack the DNA associated core proteins banded at 24 and 48.5 KD. Thisresult is in agreement with the absence of DNA in defective virus.

[0363] D) Process overview of the production and purification ofAdCMVp53 virus

[0364] Based on the above process development results, the inventorspropose a production and purification flow chart for AdCMVp53 as shownin FIG. 23. The step and accumulative virus recovery is included withthe corresponding virus yield based on a 1 mer Cellcube™. The finalvirus recovery is about 70±10%. This is about 3-fold higher than thevirus recovery reported by Huyghe et al. (1996) using a DEAE ionexchanger and a metal chelate chromatographic purification procedure forthe purification of p53 protein encoding adenovirus. Approximately3×10¹² PFU of final purified virus product was produced from a 1 merCellcube™. This represents a similar final product yield compared to thecurrent production method using double CsCl gradient ultracentrifugationfor purification.

[0365] E) Scale-up

[0366] Successful scale-up studies are have been performed with the 4mer Cellcube™ system, and are currently underway to evaluate virusproduction in the 16 mer Cellcube™ system. The crude virus solutionproduced will be filtered, concentrated and diafiltrated using a biggerPellicon cassette. The quality and recovery of the virus will bedetermined. After Benzonase treatment, the crude virus solution will bepurified using a 20 cm and a 30 cm BioProcess column for the 4 mer and16 mer, respectively.

EXAMPLE 9

[0367] Improved Ad-p53 Production in Serum-Free Suspension Culture

[0368] Adaptation of 293 cells

[0369] 293 cells were adapted to a commercially available IS293serum-free media (Irvine Scientific; Santa Ana, Calif.) by sequentiallylowering down the FBS concentration in T-flasks. The frozen cells in onevial of PDWB were thawed and placed in 10% FBS DMEM media in T-75 flaskand the cells were adapted to serum-free IS 293 media in T-flasks bylowering down the FBS concentration in the media sequentially. After 6passages in T-75 flasks the FBS% was estimated to be about 0.019%. Thecells were subcultured two more times in the T flasks before they weretransferred to spinner flasks.

[0370] Serum-free adapted 293 cells in Tflasks were adapted tosuspension culture

[0371] The above serum-free adapted cells in T-flasks were transferredto a serum-free 250 mL spinner suspension culture (100 mL workingvolume) for the suspension culture. The initial cell density was 1.18E+5vc/mL. During the cell culture the viability decreased and the bigclumps of cells were observed. After 2 more passages in T-flasks theadaptation to suspension culture was tried again. In a second attemptthe media was supplemented with heparin, at a concentration of 100 mg/L,to prevent aggregation of cells and the initial cell density wasincreased to 5.22E±5 vc/mL. During the cell culture there was someincrease of cell density and cell viability was maintained. Afterwardsthe cells were subcultured in the spinner flasks for 7 more passages andduring the passages the doubling time of the cells was progressivelyreduced and at the end of seven passages it was about 1.3 day which iscomparable to 1.2 day of the cells in 10% FBS media in the attached cellculture. In the serum-free IS 293 media supplemented with heparin almostall the cells existed as individual cells not forming aggregates ofcells in the suspension culture (Table 12). TABLE 12 Serum-FreeSuspension Culture: Adaptation to Suspension Passage No. Flask No.Average Doubling Time (days) 11 Viability decreased 13 3.4 14 3.2 15 1Viability decreased heparin added 2 4.7 3 5.0 4 3.1 16 1 5.5 2 4.8 3 4.34 4.3 17 1 2.9 2 3.5 3 2.4 4 1.7 18 1 3.5 2 13.1  3 6.1 4 3.8 19 1 2.5 22.6 3 2.3 4 2.5 20 1 1.3 (97% viability) 2 1.5 (99% viability) 3 1.8(92% viability) 4 1.3 (96% viability)

[0372] Viralproduction and growth ofcells in serum-free suspensionculture in spinnerflask

[0373] To test the production of Ad5-CMVpS3 vectors in the serun-freesuspension culture the above cells adapted to the serum-free suspensionculture were grown in 100 mL serum-free IS293 media supplemented with0.1% Pluronic F-68 and Heparin (100 mg/L) in 250 mL spinner flasks. thecells were infected at 5 MOI when the cells reached 1.36E+06 viablecells/nL on day 3. The supernatant was analyzed everyday for HPLC viralparticles/mL after the infection. No viruses were detected other thanday 3 sample. On day 3 it was 2.2E+09 vps/mL. The pfu/mL on day 6 was2.6+/−0.6E+07 pfu/mL. The per cell pfu production was estimated to be 19which is approximately 46 times below the attached culture in theserum-supplemented media. As a control the growth of cells was checkedin the absence of an infection. TABLE 13 Serum-Free Suspension Culture:Viral Production and Cell Growth Control Viral Viral w/o infection w/oinfection w/ viral media media infection exchange exchange InitialDensity 2.1 × 10⁵ 2.1 × 10⁵ 2.1 × 10⁵ (vc/mL) Cell Density at infection9.1 × 10⁵ 1.4 × 10⁶ 1.5 × 10⁶ (vc/mL) Volumetric viral production NA 2.6× 10⁷ 2.8 × 10⁸ (pfu/mL) 6 days P.I. Volumetric viral production NA NA1.3 × 10¹⁰ (HPLC vps/mL) 6 days P.I. Per cell viral production NA NA 1.3× 10⁴ (HPLC vps/cell)

[0374] Preparation ofserum-free suspension adapted 293 cell banks

[0375] As described above, after it was demonstrated the cells producethe Ad-p53 vectors, the cells were propagated in the serum-free IS293media with 0.1 % F-68 and 100 mg/L heparin in the spinner flasks to makeserum-free suspension adapted cell banks which contain 1.0E+07 viablecells/mL/vial. To collect the cells they were centrifuged down when theywere at mid-log phase growth and the viability was over 90% andresuspended in the serum-free, supplemented IS293 media and centrifugeddown again to wash out the cells. Then the cells were resuspended againin the cryopreservation media which is cold IS293 with 0.1% F-68, 100mg/L heparin, 10% DMSO and 0.1% methylcellulose resulting in IE+07viable cells/mL. The cell suspension was transferred to sterilecryopreservation vials and they were sealed and frozen in cryocontainerat −70° C. overnight. The vials were transferred to liquid nitrogenstorage. The mycoplasma test was negative.

[0376] To revive the frozen cells one vial was thawed into the 50 mLserum-free IS293 media with 0.1% F-68 and 100 mg/L heparin in a T-150.Since then the cultures were subcultured three times in 250 mL spinnerflasks. In the other study one vial was thawed into 100 mL serum-free,supplemented IS293 media in a 250 mL spinner flask. Since then thesewere subcultured in serum-free spinner flasks 2 times. In both of thestudies the cells grew very well.

[0377] Media replacement and viral production in serum-free suspensionculture in spinner flask

[0378] In the previous serurn-free viral production in the suspensionculture in the spinner flask the per cell viral production was too lowfor the serum-free suspension production to be practical. It wassupposed that this might be due to the depletion of nutrients and/or theproduction of inhibitory byproducts. To replace the spent media withfresh serum-free, supplemented IS293 media the cells were centrifugeddown on day 3 and resuspended in a fresh serum-free IS-293 mediumsupplemented with F-68 and heparin (100 mg/L) and the resulting celldensity was 1.20E+06 vc/mL and the cells were infected with Ad5-CMVp53vectors at 5 MOI. The extracellular HPLC vps/mL was 7.7E+09 vps/mL onday 3, 1.18E+10 vps/mL on day 4, 1.2E+10 vps/mL on day 5 and 1.3E+10vps/mL on day 6 and the pfu/mL on day 6 was 2.75+/−0.86E+08 tvps/mL. Theratio of HPLC viral particles to pfus was about 47. Also the cells havebeen centrifuged down and lysed with the same type of the detergentlysis buffer as used in the harvest of CellCube. The cellular HPLCvps/mL was 1.6E+10 vps/mL on day 2, 6.8E+09 vps/mL on day 3, 2.2E+09vps/mL on day 4, 2.24E+09 vps/mL on day 5 and 2.24E+09 vps/mL on day 6.

[0379] The replacement of the spent media with a fresh serum-free,supplemented IS 293 media resulted in the significant increase in theproduction of Ad-p53 vectors. The media replacement increased theproduction of extracellular HPLC viral particles 3.6 times higher abovethe previous level on day 3 and the production of extracellular pfutiter ten times higher above the previous level on day 6. Per cellproduction of Ad-p53 vectors was estimated to be approximately 1.33E+04HPLC vps.

[0380] The intracellular HPLC viral particles peaked on day 2 followingthe infection and then the particle numbers decreased. In return theextracellular viral particles increased progressively to the day 6 ofharvest. Almost all the Ad-p53 vectors were produced for the 2 daysfollowing the infection and intracellularly localized and then theviruses were released outside of the cells. Almost half of the viruseswere released outside of the cells into the supernatant between day 2and day 3 following the infection and the rate of release decreased astime goes on.

[0381] All the cells infected with Ad-p53 vectors lost their viabilityat the end of 6 days after the infection while the cells in the absenceof infection was 97% viable. In the presence of infection the pH of thespent media without the media exchange and with the media exchange was6.04 and 5.97, respectively, while the one in the absence of theinfection was 7.00 (Table 12).

[0382] Viral production and cell culture in stirred bioreactor withmedia replacement and gas overlay

[0383] To increase the production of Ad-p53 vectors, a 5L CelliGenbioreactor was used to provide a more controlled environment. In the 5 LCelliGen bioreactor the pH and the dissolved oxygen as well as thetemperature was controlled. Oxygen and carbon dioxide gas was connectedto the solenoid valve for oxygen supply and the pH adjustment,respectively. For a better mixing while generating low shear environmenta marine-blade impeller was implemented. Air was supplied all the timeduring the operation to keep a positive pressure inside the bioreactor.

[0384] To inoculate the bioreactor a vial of cells was thawed into 100mL serum-free media in a 250 mL spinner flask and the cells wereexpanded in 250 or 500 mL spinner flasks. 800 mL cell inoculum, grown in500 mL flasks, was mixed with 2700 mL fresh media in a 10 L carboy andtransferred to the CelliGen bioreactor by gas pressure. The initialworking volume of the CelliGen bioreactor was about 3.5 L culture. Theagitation speed of the marine-blade impeller was set at 80 rpm, thetemperature at 37° C., pH at 7.1 at the beginning and 7.0 after theinfection and the DO at 40% all the time during the run.

[0385] The initial cell density was 4.3E+5 vc/mL (97% viability) and 4days later when the cell density reached to 2.7E+6 vc/mL (93% viability)the cells were centrifuged down and the cells were resuspended in afresh media and transferred to the CelliGen bioreactor. After the mediaexchange the cell density was 2.1E+6 vc/mL and the cells were infectedat MOI of 10. Since then the DO dropped to below 40%. To keep the DOabove 40%, about 500 mL of culture was withdrawn from the CelliGenbioreactor to lower down the oxygen demand by the cell culture and theupper marine-blade was positioned close to the interface between the gasand the liquid phase to improve the oxygen transfer by increasing thesurface renewal. Since then the DO could be maintained above 40% untilthe end of the run.

[0386] For pH control, CO₂ gas was used to acidify the cell culture and1 N NaHCO₃ solution to make the cell culture alkaline. The pH controlwas initially set at 7.10. The initial pH of the cell culture was aboutpH 7.41. Approximately 280 mL IN NaHCO₃ solution was consumed until thepH of cell culture stabilized around pH 7.1. After the viral infectionof the cell culture, the pH control was lowered down to pH 7.0 and theCO₂ gas supply line was closed off to reduce the consumption of NaHCO₃solution. The consumption of too much NaHCO₃ solution for pH adjustmentwould increase the cell culture volume undesirably. Since then 70 mL 1NNaHCO₃ solution was consumed and the pH was in the range between 7.0 and7.1 most of the time during the run. The temperature was controlledbetween 35° C. and 37° C.

[0387] After the infection the viability of the cells decreased steadilyuntil day 6 of harvest after the infection. On the harvest day none ofthe cells was viable. The volumetric viral production of the CelliGenbioreactor was 5.1E+10 HPLC vps/mL compared to the 1.3E+10 vps/mL in thespinner flask. The controlled environment in the CelliGen bioreactorincreased the production of Ad-p53 vectors 4-fold compared to thespinner flasks with media replacement. This is both due to the increaseof the cell density at the time of infection from 1.2E+6 to 2.1E+6 vc/mLand the increase of per cell viral production from 1.3E+4 to 2.5E +4vps/mL. The 2.5E+4 vps/mL is comparable to the 3.5E+4 vps/cell in theserum-supplemented, attached cell culture.

[0388] Viral production and cell culture in stirred and spargedbioreactor

[0389] In the first study the cells were successfully grown in anstirred bioreactor for viral production, and the oxygen and C0₂ weresupplied by gas overlay in the headspace of a bioreactor. However, thismethod will limit the scale-up of the cell culture system because of itsinefficient gas transfer. Therefore in the second study, to test thefeasibility of the scale up of the serum-free suspension culture andinvestigate the growth of cells and Ad-p53 production in a spargedbioreactor, pure oxygen and C0₂ gases were supplied by bubbling throughthe serum-free IS293 media supplemented with F-68 (0.1%) and heparin(100 mg/L).

[0390] Pure oxygen was bubbled through the liquid media to supply thedissolved oxygen to the cells and the supply of pure oxygen wascontrolled by a solenoid valve to keep the dissolved oxygen above 40%.For efficient oxygen supply while minimizing the damage to the cells astainless steel sintered air diffuser, with a nominal pore size of whichis approximately 0.22 micrometer, was used for the pure oxygen delivery.The CO₂ gas was also supplied to the liquid media by bubbling from thesame diffuser and tube as the pure oxygen to maintain the pH around 7.0.For pH control Na₂CO₃ solution (106 g/L) was also hooked up to thebioreactor. Air was supplied to the head space of the bioreactor to keepa positive pressure inside the bioreactor. Other bioreactorconfiguration was the same as the first study.

[0391] Inoculum cells were developed from a frozen vial. One vial offrozen cells (1.0E+7 vc) was thawed into 50 mL media in a T-150 flaskand subcultured 3 times in 200 nL media in 500 mL spinner flasks. 400 mLof inoculun cells grown in 2 of 500 mL spinner flasks were mixed withIS293 media with F-68 and heparin in a 10 L carboy to make 3.5 L cellsuspension and it was transferred to the 5 L CelliGen bioreactor.

[0392] The initial cell density in the bioreactor was 3.0E+4 vc/mL. Theinitial cell density is lower than the first study. In the first studyfour of 500 mL spinner flasks were used as the inoculum. Even with thelower initial cell density the cells were grown up to 1.8E+6 vc/mL onday 7 in the sparged environment and the viability was 98%. During the 7days' growth, glucose concentration decreased from 5.4 g/L to 3.0 g/Land lactate increased from 0.3 g/L to 1.8 g/L.

[0393] On day 7, when the cell density reached 1.8E+6 vc/mL, the cellsin the bioreactor were centrifuged down and resuspended in 3.5 L freshserum-free IS293 media with F-68 and heparin in a 10 L carboy. The 293cells were infected with 1.25E+11 pfu Ad-p53 and transferred to theCelliGen bioreactor. In the bioreactor, cell viability was 100% but thecell density was only 7.2E+5 vc/mL. There was a loss of cells during themedia exchange operation. The viral titer in the media was measured as2.5E+10 HPLC vps/mL on day 2, 2.0E+10 on day 3, 2.8E+10 on day 4,3.5E+10 on day 5 and 3.9E+10 HPLC vps/mL on day 6 of harvest. The firstCelliGen bioreactor study with gas overlay produced 5.1E+10 HPLC vps/mL.The lower virus concentration in the second run was likely due to thelower cell density at the time of infection. Compared to the 7.2E+5vc/mL in the second run, 2.1E+6 vc/mL was used in the first run.Actually the per cell production of Ad-p53 in the second spargedCelliGen bioreactor is estimated to be 5.4E+4 vps/cell which is thehighest per cell production ever achieved so far. The per cellproduction in the first serum-free CellGen bioreactor without spargingand the serum-supplemented T-flask was 2.5E+4 vps/cell and 3.5E+4vps/cell, respectively.

[0394] After the viral infection, the viability of the cells decreasedfrom 100% to 13% on day 6 of harvest. During those 6 days after theinfection the glucose concentration decreased from 5.0 g/L to 2.1 g/Land the lactate increased from 0.3 g/L to 2.9 g/L. During the entireperiod of operation about 20 mL of Na₂CO₃ (106 g/L) solution wasconsumed.

[0395] The experimental result shows that it is technically andeconomically feasible to produce Ad-p53 in the sparged and stirredbioreactor. Scale-up and large-scale unit operation of sparged andstirred bioreactor are well established.

[0396] All of the compositions and/or methods disclosed and claimedherein can be made and executed without undue experimentation in lightof the present disclosure. While the compositions and methods of thisinvention have been described in terms of preferred embodiments, it willbe apparent to those of skill in the art that variations may be appliedto the compositions and/or methods and in the steps or in the sequenceof steps of the method described herein without departing from theconcept, spirit and scope of the invention. More specifically, it willbe apparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

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What is claimed is:
 1. A method for producing an adenovirus comprising:a) growing host cells in media at a low perfusion rate; b) infectingsaid host cells with an adenovirus; c) harvesting and lysing said hostcells to produce a crude cell lysate; d) concentrating said crude celllysate; e) exchanging buffer of crude cell lysate; and f) reducing theconcentration of contaminating nucleic acids in said crude cell lysate.2. The method of claim 1, further comprising isolating an adenoviralparticle from said cell lysate using chromatography.
 3. The method ofclaim 1, wherein the glucose concentration in said media is maintainedbetween about 0.7 and about 1.7 g/L.
 4. The method of claim 1, whereinsaid exchanging buffer involves a diafiltration step.
 5. The method ofclaim 1, wherein said adenovirus comprises an adenoviral vector encodingan exogenous gene construct.
 6. The method of claim 5, wherein said geneconstruct is operatively linked to a promoter.
 7. The method of claim 6,wherein said promoter is SV40 IE, RSV LTR, β-actin, CMV IE, adenovirusmajor late, polyoma F9-1, or tyrosinase.
 8. The method of claim 1,wherein said adenovirus is a replication-incompetent adenovirus.
 9. Themethod of claim 8, wherein the adenovirus is lacking at least a portionof the E1-region.
 10. The method of claim 9, wherein the adenovirus islacking at least a portion of the EIA and/or E1B region.
 11. The methodof claim 1, wherein said host cells are capable of complementingreplication.
 12. The method of claim 1, wherein said host cells are 293cells.
 13. The method of claim 5, wherein said exogenous gene constructencodes a therapeutic gene.
 14. The method of claim 13, wherein saidtherapeutic gene encodes antisense ras, antisense myc, antisense rafantisense erb, antisense src, antisensefins, antisense jun, antisensetrk antisense ret, antisense gsp, antisense hst, antisense bcl antisenseabl, Rb, CFTR, p16, p21, p27, p57, p73, C-CAM, APC, CTS-1, zac1, scFVras, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, BRCA1, VHL, MMAC1, FCC, MCC,BRCA2, IL-1, IL-2, IL-3, IL4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11IL-12, GM-CSF G-CSF, thymidine kinase or p53.
 15. The method of claim14, wherein said therapeutic gene encodes p53.
 16. The method of claim1, wherein said cells are harvested and lysed ex situ using a hypotonicsolution, hypertonic solution, freeze-thaw, sonication, impinging jet,microfluidization or a detergent.
 17. The method of claim 1, whereinsaid cells are harvested and lysed in situ using a hypotonic solution,hypertonic solution, or a detergent.
 18. The method of claim 17, whereinsaid cells are lysed and harvested using detergent.
 19. The method ofclaim 18, wherein said detergent is Thesit®, NP-40®, Tween-20®,Brij-58®, Triton X®-100 or octyl glucoside.
 20. The method of claim 1,wherein said lysis is achieved through autolysis of infected cells. 21.The method of claim 1, wherein said cell lysate is treated withBenzonase®, or Pulmozyme®.
 22. The method of claim 2, wherein saidisolating consists essentially of a single chromatography step.
 23. Themethod of claim 22, wherein said chromatography step is ion exchangechromatography.
 24. The method of claim 23, wherein said ion exchangechromatography is anion exchange chromatography.
 25. The method of claim24, wherein said anion exchange chromatography utilizes DEAE, TMAE, QAE,or PEI.
 26. The method of claim 24, wherein said anion exchangechromatography utilizes Toyopearl Super Q 650M, MonoQ, Source Q orFractogel TMAE.
 27. The method of claim
 24. wherein said ion exchangechromatography is carried out at a pH range of between about 7.0 andabout 10.0.
 28. The method of claim 1, further comprising aconcentration step employing membrane filtration.
 29. The method ofclaim, 28, wherein said filtration is tangential flow filtration. 30.The method of claim, 28, wherein said filtration utilizes a 100 to 300KNMWC, regenerated cellulose, or polyether sulfone membrane.
 31. Anadenovirus produced according to a process comprising the steps of: a)growing host cells in media at a low perfusion rate; b) infecting saidhost cells with an adenovirus; c) harvesting and lysing said host cellsto produce a crude cell lysate; d) concentrating said crude cell lysate;e) exchanging buffer of crude cell lysate; and f) reducing theconcentration of contaminating nucleic acids in said crude cell lysate.32. The adenovirus of claim 31, wherein adenovirus comprises anadenoviral vector encoding an exogenous gene construct.
 33. Theadenovirus of claim 31, wherein said gene construct is operativelylinked to a promoter.
 34. The adenovirus of claim 31, wherein saidadenovirus is a replication-incompetent adenovirus.
 35. The adenovirusof claim 34, wherein said adenovirus is lacking at least a portion ofthe E1-region.
 36. The adenovirus of claim 31, wherein the adenovirus islacking at least a portion of the E1A and/or E1B region.
 37. Theadenovirus of claim 31, wherein said host cells are capable ofcomplementing replication.
 38. The adenovirus of claim 31, wherein saidhost cells are 293 cells.
 39. The adenovirus of claim 31, wherein saidexogenous gene construct encodes a therapeutic gene.
 40. The adenovirusof claim 39, wherein said therapeutic gene encodes antisense ras,antisense myc, antisense raf antisense erb, antisense src, antisensefis, antisense jun, antisense trk antisense ret, antisense gsp,antisense hst, antisense bcl antisense abl, Rb, CFTR, p16, p21, p27,p57, p73, C-CAM, APC, CTS-1, zac1, scFV ras, DCC, NF-1, NF-2, WT-1,MEN-I, MEN-II, BRCA1, VHL, MMAC1, FCC, MCC, BRCA2, IL-1, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, GM-CSF G-CSF,thyrnidine kinase or p53.
 41. The adenovirus of claim 40, wherein saidtherapeutic gene is p53.
 42. The adenovirus of claim 33, wherein saidpromoter is SV40 IE, RSV LTR, β-actin or CMV IE, adenovirus major late,polyoma F9-1, or tyrosinase.
 43. A method for the purification of anadenovirus comprising: a) growing host cells; b) infecting said hostcells with an adenovirus; c) harvesting and lysing said host cells bycontacting said cells with a detergent to produce a crude cell lysate;d) concentrating said crude cell lysate; e) exchanging buffer of crudecell lysate; and f) reducing the concentration of contaminating nucleicacids in said crude cell lysate.
 44. The method of claim 43, furthercomprising isolating an adenoviral particle from said lysate usingchromatography.
 45. The method of claim 43, wherein said host cells aregrown in media wherein a glucose concentration is maintained betweenabout 0.7 and about 1.7 g/L.
 46. The method of claim 43, wherein saidexchanging buffer involves a diafiltration step.
 47. The method of claim43, wherein said detergent is Thesit®, NP-40®, Tween-20®, Brij-58®,Triton X-100 or octyl glucoside.
 48. The method of claim 47, whereinsaid detergent is present in the lysis solution at a concentration ofabout 1% (w/v).
 49. The method of claim 43, wherein said isolatingconsists essentially of a single chromatography step.
 50. The method ofclaim 44, wherein said chromatography step is ion exchangechromatography.
 51. An adenovirus produced according to a processcomprising the steps of: a) growing host cells; b) infecting said hostcells with an adenovirus; c) harvesting and lysing said host cells bycontacting said cells with a detergent to produce a crude cell lysate;d) concentrating said crude cell lysate; e) exchanging buffer of crudecell lysate; and f) reducing the concentration of contaminating nucleicacids in said crude cell lysate.
 52. A method for the purification of anadenovirus comprising: a) growing host cells in serum-free media; b)infecting said host cells with an adenovirus; c) harvesting and lysingsaid host cells to produce a crude cell lysate; d) concentrating saidcrude cell lysate; e) exchanging buffer of crude cell lysate; and f)reducing the concentration of contaminating nucleic acids in said crudecell lysate.
 53. The method of claim 52, wherein said host cells areadapted for growth in serum-free media.
 54. The method of claim 52,wherein said cells are grown as a cell suspension culture.
 55. Themethod of claim 52, wherein said cells are grown as ananchorage-dependent culture.
 56. The method of claim 53, wherein saidadaptation for growth in serum-free media comprises a sequentialdecrease in the fetal bovine serum content of the growth media.
 57. Themethod of claim 53, wherein said serum-free media comprises a fetalbovine serum content of less than 0.03% v/v.
 58. The method of claim 52,further comprising isolating an adenoviral particle from said lysateusing chromatography.
 59. The method of claim 52, wherein said lysis isachieved through autolysis of infected cells.
 60. The method of claim52, wherein said exchanging buffer involves a diafiltration step. 61.The method of claim 52, wherein said detergent is Thesit®, NP-40®,Tween-20®, Brij-58®, Triton X-100® or octyl glucoside.
 62. The method ofclaim 52, wherein said detergent is present in the lysis solution at aconcentration of about 1% (w/v).
 63. The method of claim 52, whereinsaid isolating consists essentially of a single chromatography step. 64.The method of claim 58, wherein said chromatography step is ion exchangechromatography.
 65. An adenovirus produced according to a processcomprising the steps of: a) growing host cells in serum-free media; b)infecting said host cells with an adenovirus; c) harvesting and lysingsaid host cells to produce a crude cell lysate; d) concentrating saidcrude cell lysate; e) exchanging buffer of crude cell lysate; and f)reducing the concentration of contaminating nucleic acids in said crudecell lysate.
 66. A 293 host cell adapted for growth in serum-free media.67. The cell of claim 66, wherein said cell is adapted for growth insuspension culture.
 68. The cell of claim 66, wherein the cell isdeposited with the ATCC and is designated as a IT293SF cell.
 69. Thecell of claim 66, wherein said adaptation for growth in serum-free mediacomprises a sequential decrease in the fetal bovine serum content of thegrowth media.”